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Cite this: Analyst, 2014, 139, 1830
Received 2nd January 2014 Accepted 24th January 2014
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Versatile chiral chromatography with mixed stationary phases of water-impregnated silica gel and reversed-phase packing† Satsuki Takahashi and Tetsuo Okada*
DOI: 10.1039/c4an00003j www.rsc.org/analyst
A novel chiral chromatographic scheme is proposed, which requires no organic syntheses in stationary phase preparation. A normal phase chiral chromatography stationary phase is prepared by simple passage of an aqueous solution of an appropriate chiral selector through e.g. a silica gel column. For this scheme, a mixed-bed of bare silica gel and octadecylsilanized silica gel (ODS) provides much better separation performance than a silica gel column. Isolation of silica gel particles in the column is important for successful chiral separation based on the present scheme.
Introduction Chiral liquid chromatographic separation is oen performed with a stationary phase, on which a selector (CS) is chemically bonded.1–7 Polysaccharides including cellulose,4,5 cyclodextrin and its derivatives,6 protein,7 etc. have been successfully employed as CSs for chiral chromatography. Since the applicability of CSs is usually limited to a particular class of enantiomers, the stationary phase should be carefully selected to attain required separation; i.e. a given stationary phase may be effective for chiral separation of particular compounds but may not work at all for separating other enantiomers.8,9 It should therefore be very useful if we can introduce a given CS into a common stationary phase in a simple way that requires no heavy organic syntheses. We reported chiral ice chromatography, in which an ice containing CS was used as a chiral liquid chromatographic stationary phase.10 An advantage of this method over usual chiral chromatography is that the stationary phase can be prepared by simple freezing of an aqueous solution of CSs and, thus, no organic syntheses are necessary for stationary phase preparation. Some enantiomers have been successfully separated with the Department of Chemistry, Tokyo Institute of Technology, Meguro-ku, Tokyo 152-8551, Japan. E-mail: [email protected]; Fax: +81-3-5734-2612; Tel: +81-3-57342612 † Electronic supplementary information (ESI) available: Temperature dependent chiral separation of hexobarbital and bisnaphthol, DSC of water in silica gel pores, and chiral chromatograms with various CSs. See DOI: 10.1039/c4an00003j
1830 | Analyst, 2014, 139, 1830–1833
b-cyclodextrin (bCD)-incorporated ice stationary phase. The ice stationary phase has, however, intrinsic analytical disadvantages, e.g. poor separation efficiency and limited durability, which hinder the wide applications of this method.11,12 Water molecules on the surface of ice have rather high mobility, which causes sintering of ice particles. The effective surface area of the ice stationary phase is rapidly diminished thereby, and separation also becomes poorer. Column storage suffers from the same problem. Even though the column is stored at low temperature, sintering proceeds and the column efficiency becomes lower during storage. Chiral recognition of bCD in the ice stationary phase occurs not on the surface of ice but in the liquid phase coexistent with ice.10 If the liquid phase can be stably retained in the stationary phase, a solid support does not have to be ice. Porous silica gel can, for example, be used for this purpose. However, it has been found that water-impregnation in silica gel pores results in unexpectedly poor separation, and, therefore, chiral separation has not been conrmed with such stationary phases. In the present communication, we propose a new idea to dramatically improve the separation efficiency of water-impregnated silica gel stationary phase and report chiral separation with the stationary phase prepared in this simple and versatile way.
Experimental section The chromatographic system was composed of a Shimadzu degasser unit Model DGU-20A, a Shimadzu HPLC pump Model LC30AD, a Rheodyne injection valve equipped with a 20 mL sample loop, a JASCO circular dichroism detector Model CD2095 Plus, and a Huber low-temperature bath ModelCC-410wl. The stationary phases were Daiso SP-1000-10 silica gel (10 mm in diameter, mean pore size 100 nm), Wakosil 10SIL (silica gel, 10 mm in diameter, mean pore size 6 nm), and Wakosil 10C18 (10 mm in diameter). The stationary phase was packed in a stainless HPLC column (4.6 mm i.d. 75 mm) by the slurry packing method.13 An aqueous solution containing CS (ca. 100 mL) passed through the column, and then the column was
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rinsed with hexane. This procedure allowed the replacement of the aqueous solution in the extra particle space, but the aqueous phase remained in the pores of the stationary phase. The mobile phase was hexane containing THF or chloroform. The amount of an aqueous solution retained in the stationary phase was determined by the Karl-Fischer titration of column effluents rinsed by isopropanol with a Metrohm Model 870 KF Titrino Plus. For chromatographic measurements under frozen conditions, the water-impregnated column was immersed in partially frozen acetonitrile (42 C) and then the temperature was raised to the operation point. Differential scanning calorimetric (DSC) measurements were conducted with a DSC Perkin Elmer Model DSC8500 equipped with a cooling unit.
Results and discussion bCD forms an inclusion complex in an aqueous phase by accommodating a guest molecule in its hydrophobic cavity. Chiral recognition with bCD should therefore occur in an aqueous bCD solution.14 In chiral ice chromatography, a salt was added to the ice stationary phase containing bCD to allow the development of an aqueous liquid phase supported by the ice matrix. The simplest way to retain an aqueous solution in the stationary phase is its impregnation in the pores of a solid support, such as silica gel. This is the same scenario as the rst partition liquid chromatography invented by Martin and Synge.15 However, we have found that simple water-impregnation in silica gel leads to poor separation. Very weak retention was for example conrmed for hexobarbital with silica gel containing an aqueous bCD solution as shown in Fig. 1A. Bulk extraction experiments have revealed the moderate partition of this analyte to an aqueous phase (Kd ¼ 0.2–0.5, depending on the salt concentration in an aqueous phase).10 If the entire separation system is in equilibrium, the retention of hexobarbital should be higher, suggesting that all of the aqueous phase does not act for solute partition. Severe peak broadening occurred for 1,10 -bis-2-naphthol (bisN) even when the peak appeared immediately aer the unretained peak as shown in Fig. S1;† the theoretical plate number was smaller than 15 for this solute. The poor separation efficiency can be interpreted assuming the wide-range connection of the aqueous phase in the extra particle space as schematically illustrated in Fig. 2A. The formation of a massive aqueous phase in the column hinders the effective solute diffusion in the mobile phase, reduces the effective interfacial area, and causes multi-isolated streamlines that are mutually not accessible. It is thus important to prevent aqueous solutions impregnated in silica gel pores from forming a wide-range network in order to attain better separation based on the present idea. We here propose the utilization of a mixed column bed, in which stationary phase beads of entirely different surface properties are incorporated. Since water-impregnated silica gel beads are highly hydrophilic, the contact of the beads causes the water connection in the extra particle space. The incorporation of octadecylsilanized silica gel (ODS) beads is expected to allow the connement of aqueous solution near silica beads (Fig. 2B). Even though the aqueous solution partially came out of the
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Fig. 1 Chiral separation of hexobarbital. Column, 4.6 mm i.d. 75 mm. Stationary phase, 1 mM bCD + 75 mM KCl impregnated (A) silica gel, (B) 33% silica gel/ODS and (C) 25% silica gel/ODS. Mobile phase, 3% THF in hexane. Temperature, 20 C.
pores, it should stay in the vicinity of the silica gel beads and would not be spread over a wide range. Fig. 1 demonstrates the successful chiral separation of the hexobarbital enantiomers based on the present idea; mixed stationary phases of 1 : 2 and 1 : 3 (silica : ODS) containing bCD as CS were employed. Although the 1 : 3 stationary phase gives smaller retention due to the reduction of the aqueous phase volume supported in the pores of silica gel, the enantiomers are separated well; the theoretical plate number increased up to ca. 500, corresponding to ca. 1000 for a 15 cm long usual column. In the present case, the retention factor (k) of an analyte is given by10 Kd Vwater 1 þ K1 ½CD þ K1 K2 ½CD2 k ¼ kads þ Vmob where kad represents the retention by the adsorption of a solute, Kd (¼[s]water /[s]mob, the ratio of the solute concentrations in the water phase to that in the mobile phase) is the partition
Fig. 2 Schematic representations of water-impregnated silica gel beads and their spatial isolation by mixed packing with ODS beads.
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coefficient of the solute, [CD] is the concentration of bCD in the water phase, Vwater and Vmob are the volumes of the water and mobile phases in the column, and K1 and K2 are the association constants for 1 : 1 (s-CD) and 1 : 2 (s-CD2) complexes between a solute (s) and bCD, respectively. For an analyte forming only a 1 : 1 complex, the last term in the right side of the above equation can be omitted. Fig. 3 shows the amount of water retained in a 4.6 75 mm column as a function of a weight ratio of silica gel in the mixed silica/ODS stationary phase. The water amount linearly decreases with decreasing silica gel ratio in the stationary phase. The amount of water retained in the ODS stationary phase is ca. onesixth as small as that in silica gel. The aqueous phase is thus mostly retained in silica gel. The above equation suggests that the retention factor depends on Vwater at constant [CD] as long as kad is constant. A change in the amount of the ODS beads packed in the column may cause a variation of kad. However, it has been conrmed that the adsorption of a solute on the ODS stationary phase is so weak in the present mobile phase condition that ODS is expected to act as the inert spacer to keep silica gel beads not in contact with one another. A decrease in the silica gel fraction in the column packing reduces Vwater and, in turn, solute retention. Therefore, the 25% silica gel column gives lower retention than the 33% counterpart as shown in Fig. 1. Temperature is also an important parameter governing chiral separation. Fig. 4 and 5 show the temperature dependence of various separation parameters obtained for the enantioseparation of hexobarbital and bisN, respectively. Silica gel with large pores (Daiso SP) was employed to examine freezing effects on separation parameters such as the separation ratio (a) and resolution (Rs). Chromatograms measured at various temperatures are shown in Fig. S2 and S3.† In both compounds, the retention increases with decreasing temperature due to the exothermic nature of bCD complexation. A difference in the retention time between hexobarbital enantiomers becomes larger as the temperature decreases; interestingly, the temperature dependence of the bCD complexation of one enantiomer appears very small as shown in Fig. 4. A separation ratio for hexobarbital enantiomers increases from 1.7 at 20 C to 3.0 at 15 C as a result. However, lowering the temperature simultaneously reduces the diffusivity of an analyte and leads to band
Fig. 3 Change in the amount of water retained in stationary phase pores with the weight ratio of silica gel to the entire column packing.
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Fig. 4 Temperature dependence of (A) retention times and (B) separation ratio, a, and resolution, Rs, for the enantiomers of hexobarbital. Column, 4.6 mm i.d. 75 mm. Stationary phase, 1 mM bCD + 75 mM KCl impregnated 33% Daiso SP/ODS. Mobile phase, 3% THF in hexane.
broadening. Thus, the temperature affects the separation in two opposite ways, i.e. a difference in retention increases but peak broadening becomes severe with decreasing temperature. The maximum resolution is therefore produced in the middle temperature range around 0 C. In contrast, the separation ratio for bisN only slightly increases with decreasing temperature, and the resolution does not show clear temperature dependence. However, it should be noted that the enantiomers of bisN are not separated at room temperature with this stationary phase as shown in Fig. S2.† DSC measurements have indicated that water in the pores of Daiso SP silica gel is frozen at ca. 20 C; the melting point of formed ice is ca. 2 C (Fig. S4†). It is known that the melting
Fig. 5 Temperature dependence of (A) retention times and (B) separation ratio, a, and resolution, Rs, for the enantiomers of bisN. Column, 4.6 mm i.d. 75 mm. Stationary phase, 1 mM bCD + 75 mM KCl impregnated 33% Daiso SP/ODS. Mobile phase, 1% THF in hexane.
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point of water in a small space is lowered and the melting point depression (DT) can be explained by the Gibbs–Thomson effect.16
Published on 24 January 2014. Downloaded by Queens University - Kingston on 26/10/2014 10:31:48.
DT ¼
2T0 gSL VL DHr
where T0 is the melting point (273 K), gSL is the interfacial tension between ice and liquid water (ca. 31.7 mJ m2), VL is the molar volume of liquid water (1.8 105 m3 mol1), DH is the melting enthalpy of ice (6.01 kJ mol1), and r is the radius of the space, in which water is conned, respectively. According to this equation, the melting point of ice is lowered by ca. 1 K in r ¼ 50 nm (in Daiso SP silica gel) and 17 K in the 6 nm pores in Wakosil 10SIL. As described in the Experimental section, the stationary phase was frozen at 42 C and the temperature was raised to the operation point for low temperature operations. Thus, water in the pore of Daiso SP silica gel remains frozen at a temperature lower than 2 C. It is not clear whether the presence of ice is favorable or unfavorable for chiral separation because a clear discontinuity is not found in any retention data in Fig. 4 and 5. However, the temperature is still an important experimental parameter and should be optimized for desired separation. In addition, from a practical point of view, the low temperature operation is advantageous because the column durability is enhanced. The aqueous phase retained in the silica gel pores is likely to be lost during chromatographic operation for a long time period, reducing the separation performance. This column degradation is largely improved by low temperature operation. The column can be repeatedly used over several days. The present scheme was applied to other water-soluble CSs. Table 1 summarizes selected separation data obtained with bCD, 3-hydroxyl-propyl bCD, or L-proline + bCD as CSs at the room temperature. The corresponding chromatograms are shown in Fig. S5 and S6 in the ESI.† Since low temperature is not necessary for efficient chiral separation with the present scheme, silica gel with small pores (Wakosil 10SIL) was employed with ODS. As shown above, though bCD was effective for separation of the enantiomers of hexobarbital and bisN, these were not separated with hydroxyl-propyl bCD. In contrast, bCD did not separate the enantiomers of 3-hydroxy avonon, which were resolved with OHPrbCD though base-line separation was not attained. Thus, the separation selectivity of the stationary phase can easily be varied by changing a loaded CS. The same packed column can be repeatedly used for the preparation of different stationary phases in the present scheme.
Table 1 Selected separation data with a 1 : 2 Wakosil 10SIL-ODS mixed stationary phase impregnated with various CSs
CS
Analyte
a
Rs
Mobile phasea
bCDe
2HFlb Ibuprofen 2HFl 3HFlc BisNd
1.39 1.17 1.22 1.11 1.43
2.38 0.88 1.58 0.98 2.67
3% THF
OHPrbCD f Pro g
A mixed silica gel and ODS column bed has allowed us to prepare chiral stationary phases in a versatile and simple way. This approach has high applicability, i.e. various chiral stationary phases can be prepared without anchoring CSs on a solid support. Also, a similar approach can be used for lipophilic CSs, which can be impregnated in the pores of ODS. Dynamic coating has been known as an effective way to prepare chiral stationary phases.17,18 One of the important differences between dynamic coating and our method is that a partition process is involved in the separation mechanism in the present case. We can manage an extent of the solute partition by changing various experimental parameters such as the volume of the liquid phase, temperature, pH, ionic strength, and type of solvent. Also, the concentration of CS can be changed in the present method, though it is difficult in dynamic coating chromatography to vary the CS concentration.
Acknowledgements This work has been supported by SENTAN from the Japan Science and Technology Agency.
Notes and references 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
3% THF 10% CHCl3
Mobile phases were prepared in hexane. 2-Hydroxy avonon. 3-Hydroxy avonon. d 1,10 -Bis-2-naphthol. e 10 mM bCD. f 10 mM 3-hydroxyl-propyl bCD. g 10 mM L-proline containing bCD. a
Conclusions
17
b
c
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18
R. Healey and A. Ghanem, Chirality, 2013, 25, 314–323. H. Lu and G. Chen, Anal. Methods, 2011, 3, 488–508. B. Kasprzyk-Hordern, Chem. Soc. Rev., 2010, 39, 4466–4503. Y. Okamoto and Y. Kaida, J. Chromatogr. A, 1994, 666, 403– 419. G. M. Peng, S. Q. Wu, W. G. Zhang, J. Fan, S. R. Zhang, G. Xu and X. Q. Xu, Anal. Sci., 2013, 29, 637–642. Z. Juvancz and J. Szejtli, TrAC, Trends Anal. Chem., 2002, 21, 379–388. J. Haginaka, J. Chromatogr. B: Anal. Technol. Biomed. Life Sci., 2008, 875, 12–19. H. Y. Aboul-Enein and I. Ali, Chromatographia, 2000, 52, 679– 691. C. V. Hoffmann, R. Pell and M. Laemmerhofer, Anal. Chem., 2008, 80, 8780–8789. T. Shamoto, Y. Tasaki and T. Okada, J. Am. Chem. Soc., 2010, 132, 13135–13137. Y. Tasaki and T. Okada, Anal. Chem., 2006, 78, 4155–4160. Y. Tasaki and T. Okada, Anal. Chem., 2009, 81, 890–897. P. R. Bristow, P. N. Brittain, C. M. Riley and B. F. Williams, J. Chromatogr., 1977, 131, 57–64. K. A. Connors, Chem. Rev., 1997, 97, 1325–1357. A. J. P. Martin and R. L. M. Synge, Biochem. J., 1941, 35, 91. S. J¨ ahnert, F. V. Ch´ avez, G. E. Shaumann, A. Schreiber, M. Sch¨ onhoff and G. H. Findenegg, Phys. Chem. Chem. Phys., 2008, 10, 6039–6051. T. Shinbo, T. Yamaguchi, K. Nishimura and M. Sugiura, J. Chromatogr., 1987, 405, 145–153. M. H. Hyun, H. J. Koo, J. S. Jin and W. Lee, J. Liq. Chromatogr. Relat. Technol., 2000, 23, 2669–2682. Analyst, 2014, 139, 1830–1833 | 1833
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COMMUNICATION
Cite this: Analyst, 2014, 139, 1830
Received 2nd January 2014 Accepted 24th January 2014
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Versatile chiral chromatography with mixed stationary phases of water-impregnated silica gel and reversed-phase packing† Satsuki Takahashi and Tetsuo Okada*
DOI: 10.1039/c4an00003j www.rsc.org/analyst
A novel chiral chromatographic scheme is proposed, which requires no organic syntheses in stationary phase preparation. A normal phase chiral chromatography stationary phase is prepared by simple passage of an aqueous solution of an appropriate chiral selector through e.g. a silica gel column. For this scheme, a mixed-bed of bare silica gel and octadecylsilanized silica gel (ODS) provides much better separation performance than a silica gel column. Isolation of silica gel particles in the column is important for successful chiral separation based on the present scheme.
Introduction Chiral liquid chromatographic separation is oen performed with a stationary phase, on which a selector (CS) is chemically bonded.1–7 Polysaccharides including cellulose,4,5 cyclodextrin and its derivatives,6 protein,7 etc. have been successfully employed as CSs for chiral chromatography. Since the applicability of CSs is usually limited to a particular class of enantiomers, the stationary phase should be carefully selected to attain required separation; i.e. a given stationary phase may be effective for chiral separation of particular compounds but may not work at all for separating other enantiomers.8,9 It should therefore be very useful if we can introduce a given CS into a common stationary phase in a simple way that requires no heavy organic syntheses. We reported chiral ice chromatography, in which an ice containing CS was used as a chiral liquid chromatographic stationary phase.10 An advantage of this method over usual chiral chromatography is that the stationary phase can be prepared by simple freezing of an aqueous solution of CSs and, thus, no organic syntheses are necessary for stationary phase preparation. Some enantiomers have been successfully separated with the Department of Chemistry, Tokyo Institute of Technology, Meguro-ku, Tokyo 152-8551, Japan. E-mail: [email protected]; Fax: +81-3-5734-2612; Tel: +81-3-57342612 † Electronic supplementary information (ESI) available: Temperature dependent chiral separation of hexobarbital and bisnaphthol, DSC of water in silica gel pores, and chiral chromatograms with various CSs. See DOI: 10.1039/c4an00003j
1830 | Analyst, 2014, 139, 1830–1833
b-cyclodextrin (bCD)-incorporated ice stationary phase. The ice stationary phase has, however, intrinsic analytical disadvantages, e.g. poor separation efficiency and limited durability, which hinder the wide applications of this method.11,12 Water molecules on the surface of ice have rather high mobility, which causes sintering of ice particles. The effective surface area of the ice stationary phase is rapidly diminished thereby, and separation also becomes poorer. Column storage suffers from the same problem. Even though the column is stored at low temperature, sintering proceeds and the column efficiency becomes lower during storage. Chiral recognition of bCD in the ice stationary phase occurs not on the surface of ice but in the liquid phase coexistent with ice.10 If the liquid phase can be stably retained in the stationary phase, a solid support does not have to be ice. Porous silica gel can, for example, be used for this purpose. However, it has been found that water-impregnation in silica gel pores results in unexpectedly poor separation, and, therefore, chiral separation has not been conrmed with such stationary phases. In the present communication, we propose a new idea to dramatically improve the separation efficiency of water-impregnated silica gel stationary phase and report chiral separation with the stationary phase prepared in this simple and versatile way.
Experimental section The chromatographic system was composed of a Shimadzu degasser unit Model DGU-20A, a Shimadzu HPLC pump Model LC30AD, a Rheodyne injection valve equipped with a 20 mL sample loop, a JASCO circular dichroism detector Model CD2095 Plus, and a Huber low-temperature bath ModelCC-410wl. The stationary phases were Daiso SP-1000-10 silica gel (10 mm in diameter, mean pore size 100 nm), Wakosil 10SIL (silica gel, 10 mm in diameter, mean pore size 6 nm), and Wakosil 10C18 (10 mm in diameter). The stationary phase was packed in a stainless HPLC column (4.6 mm i.d. 75 mm) by the slurry packing method.13 An aqueous solution containing CS (ca. 100 mL) passed through the column, and then the column was
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rinsed with hexane. This procedure allowed the replacement of the aqueous solution in the extra particle space, but the aqueous phase remained in the pores of the stationary phase. The mobile phase was hexane containing THF or chloroform. The amount of an aqueous solution retained in the stationary phase was determined by the Karl-Fischer titration of column effluents rinsed by isopropanol with a Metrohm Model 870 KF Titrino Plus. For chromatographic measurements under frozen conditions, the water-impregnated column was immersed in partially frozen acetonitrile (42 C) and then the temperature was raised to the operation point. Differential scanning calorimetric (DSC) measurements were conducted with a DSC Perkin Elmer Model DSC8500 equipped with a cooling unit.
Results and discussion bCD forms an inclusion complex in an aqueous phase by accommodating a guest molecule in its hydrophobic cavity. Chiral recognition with bCD should therefore occur in an aqueous bCD solution.14 In chiral ice chromatography, a salt was added to the ice stationary phase containing bCD to allow the development of an aqueous liquid phase supported by the ice matrix. The simplest way to retain an aqueous solution in the stationary phase is its impregnation in the pores of a solid support, such as silica gel. This is the same scenario as the rst partition liquid chromatography invented by Martin and Synge.15 However, we have found that simple water-impregnation in silica gel leads to poor separation. Very weak retention was for example conrmed for hexobarbital with silica gel containing an aqueous bCD solution as shown in Fig. 1A. Bulk extraction experiments have revealed the moderate partition of this analyte to an aqueous phase (Kd ¼ 0.2–0.5, depending on the salt concentration in an aqueous phase).10 If the entire separation system is in equilibrium, the retention of hexobarbital should be higher, suggesting that all of the aqueous phase does not act for solute partition. Severe peak broadening occurred for 1,10 -bis-2-naphthol (bisN) even when the peak appeared immediately aer the unretained peak as shown in Fig. S1;† the theoretical plate number was smaller than 15 for this solute. The poor separation efficiency can be interpreted assuming the wide-range connection of the aqueous phase in the extra particle space as schematically illustrated in Fig. 2A. The formation of a massive aqueous phase in the column hinders the effective solute diffusion in the mobile phase, reduces the effective interfacial area, and causes multi-isolated streamlines that are mutually not accessible. It is thus important to prevent aqueous solutions impregnated in silica gel pores from forming a wide-range network in order to attain better separation based on the present idea. We here propose the utilization of a mixed column bed, in which stationary phase beads of entirely different surface properties are incorporated. Since water-impregnated silica gel beads are highly hydrophilic, the contact of the beads causes the water connection in the extra particle space. The incorporation of octadecylsilanized silica gel (ODS) beads is expected to allow the connement of aqueous solution near silica beads (Fig. 2B). Even though the aqueous solution partially came out of the
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Fig. 1 Chiral separation of hexobarbital. Column, 4.6 mm i.d. 75 mm. Stationary phase, 1 mM bCD + 75 mM KCl impregnated (A) silica gel, (B) 33% silica gel/ODS and (C) 25% silica gel/ODS. Mobile phase, 3% THF in hexane. Temperature, 20 C.
pores, it should stay in the vicinity of the silica gel beads and would not be spread over a wide range. Fig. 1 demonstrates the successful chiral separation of the hexobarbital enantiomers based on the present idea; mixed stationary phases of 1 : 2 and 1 : 3 (silica : ODS) containing bCD as CS were employed. Although the 1 : 3 stationary phase gives smaller retention due to the reduction of the aqueous phase volume supported in the pores of silica gel, the enantiomers are separated well; the theoretical plate number increased up to ca. 500, corresponding to ca. 1000 for a 15 cm long usual column. In the present case, the retention factor (k) of an analyte is given by10 Kd Vwater 1 þ K1 ½CD þ K1 K2 ½CD2 k ¼ kads þ Vmob where kad represents the retention by the adsorption of a solute, Kd (¼[s]water /[s]mob, the ratio of the solute concentrations in the water phase to that in the mobile phase) is the partition
Fig. 2 Schematic representations of water-impregnated silica gel beads and their spatial isolation by mixed packing with ODS beads.
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coefficient of the solute, [CD] is the concentration of bCD in the water phase, Vwater and Vmob are the volumes of the water and mobile phases in the column, and K1 and K2 are the association constants for 1 : 1 (s-CD) and 1 : 2 (s-CD2) complexes between a solute (s) and bCD, respectively. For an analyte forming only a 1 : 1 complex, the last term in the right side of the above equation can be omitted. Fig. 3 shows the amount of water retained in a 4.6 75 mm column as a function of a weight ratio of silica gel in the mixed silica/ODS stationary phase. The water amount linearly decreases with decreasing silica gel ratio in the stationary phase. The amount of water retained in the ODS stationary phase is ca. onesixth as small as that in silica gel. The aqueous phase is thus mostly retained in silica gel. The above equation suggests that the retention factor depends on Vwater at constant [CD] as long as kad is constant. A change in the amount of the ODS beads packed in the column may cause a variation of kad. However, it has been conrmed that the adsorption of a solute on the ODS stationary phase is so weak in the present mobile phase condition that ODS is expected to act as the inert spacer to keep silica gel beads not in contact with one another. A decrease in the silica gel fraction in the column packing reduces Vwater and, in turn, solute retention. Therefore, the 25% silica gel column gives lower retention than the 33% counterpart as shown in Fig. 1. Temperature is also an important parameter governing chiral separation. Fig. 4 and 5 show the temperature dependence of various separation parameters obtained for the enantioseparation of hexobarbital and bisN, respectively. Silica gel with large pores (Daiso SP) was employed to examine freezing effects on separation parameters such as the separation ratio (a) and resolution (Rs). Chromatograms measured at various temperatures are shown in Fig. S2 and S3.† In both compounds, the retention increases with decreasing temperature due to the exothermic nature of bCD complexation. A difference in the retention time between hexobarbital enantiomers becomes larger as the temperature decreases; interestingly, the temperature dependence of the bCD complexation of one enantiomer appears very small as shown in Fig. 4. A separation ratio for hexobarbital enantiomers increases from 1.7 at 20 C to 3.0 at 15 C as a result. However, lowering the temperature simultaneously reduces the diffusivity of an analyte and leads to band
Fig. 3 Change in the amount of water retained in stationary phase pores with the weight ratio of silica gel to the entire column packing.
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Fig. 4 Temperature dependence of (A) retention times and (B) separation ratio, a, and resolution, Rs, for the enantiomers of hexobarbital. Column, 4.6 mm i.d. 75 mm. Stationary phase, 1 mM bCD + 75 mM KCl impregnated 33% Daiso SP/ODS. Mobile phase, 3% THF in hexane.
broadening. Thus, the temperature affects the separation in two opposite ways, i.e. a difference in retention increases but peak broadening becomes severe with decreasing temperature. The maximum resolution is therefore produced in the middle temperature range around 0 C. In contrast, the separation ratio for bisN only slightly increases with decreasing temperature, and the resolution does not show clear temperature dependence. However, it should be noted that the enantiomers of bisN are not separated at room temperature with this stationary phase as shown in Fig. S2.† DSC measurements have indicated that water in the pores of Daiso SP silica gel is frozen at ca. 20 C; the melting point of formed ice is ca. 2 C (Fig. S4†). It is known that the melting
Fig. 5 Temperature dependence of (A) retention times and (B) separation ratio, a, and resolution, Rs, for the enantiomers of bisN. Column, 4.6 mm i.d. 75 mm. Stationary phase, 1 mM bCD + 75 mM KCl impregnated 33% Daiso SP/ODS. Mobile phase, 1% THF in hexane.
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point of water in a small space is lowered and the melting point depression (DT) can be explained by the Gibbs–Thomson effect.16
Published on 24 January 2014. Downloaded by Queens University - Kingston on 26/10/2014 10:31:48.
DT ¼
2T0 gSL VL DHr
where T0 is the melting point (273 K), gSL is the interfacial tension between ice and liquid water (ca. 31.7 mJ m2), VL is the molar volume of liquid water (1.8 105 m3 mol1), DH is the melting enthalpy of ice (6.01 kJ mol1), and r is the radius of the space, in which water is conned, respectively. According to this equation, the melting point of ice is lowered by ca. 1 K in r ¼ 50 nm (in Daiso SP silica gel) and 17 K in the 6 nm pores in Wakosil 10SIL. As described in the Experimental section, the stationary phase was frozen at 42 C and the temperature was raised to the operation point for low temperature operations. Thus, water in the pore of Daiso SP silica gel remains frozen at a temperature lower than 2 C. It is not clear whether the presence of ice is favorable or unfavorable for chiral separation because a clear discontinuity is not found in any retention data in Fig. 4 and 5. However, the temperature is still an important experimental parameter and should be optimized for desired separation. In addition, from a practical point of view, the low temperature operation is advantageous because the column durability is enhanced. The aqueous phase retained in the silica gel pores is likely to be lost during chromatographic operation for a long time period, reducing the separation performance. This column degradation is largely improved by low temperature operation. The column can be repeatedly used over several days. The present scheme was applied to other water-soluble CSs. Table 1 summarizes selected separation data obtained with bCD, 3-hydroxyl-propyl bCD, or L-proline + bCD as CSs at the room temperature. The corresponding chromatograms are shown in Fig. S5 and S6 in the ESI.† Since low temperature is not necessary for efficient chiral separation with the present scheme, silica gel with small pores (Wakosil 10SIL) was employed with ODS. As shown above, though bCD was effective for separation of the enantiomers of hexobarbital and bisN, these were not separated with hydroxyl-propyl bCD. In contrast, bCD did not separate the enantiomers of 3-hydroxy avonon, which were resolved with OHPrbCD though base-line separation was not attained. Thus, the separation selectivity of the stationary phase can easily be varied by changing a loaded CS. The same packed column can be repeatedly used for the preparation of different stationary phases in the present scheme.
Table 1 Selected separation data with a 1 : 2 Wakosil 10SIL-ODS mixed stationary phase impregnated with various CSs
CS
Analyte
a
Rs
Mobile phasea
bCDe
2HFlb Ibuprofen 2HFl 3HFlc BisNd
1.39 1.17 1.22 1.11 1.43
2.38 0.88 1.58 0.98 2.67
3% THF
OHPrbCD f Pro g
A mixed silica gel and ODS column bed has allowed us to prepare chiral stationary phases in a versatile and simple way. This approach has high applicability, i.e. various chiral stationary phases can be prepared without anchoring CSs on a solid support. Also, a similar approach can be used for lipophilic CSs, which can be impregnated in the pores of ODS. Dynamic coating has been known as an effective way to prepare chiral stationary phases.17,18 One of the important differences between dynamic coating and our method is that a partition process is involved in the separation mechanism in the present case. We can manage an extent of the solute partition by changing various experimental parameters such as the volume of the liquid phase, temperature, pH, ionic strength, and type of solvent. Also, the concentration of CS can be changed in the present method, though it is difficult in dynamic coating chromatography to vary the CS concentration.
Acknowledgements This work has been supported by SENTAN from the Japan Science and Technology Agency.
Notes and references 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
3% THF 10% CHCl3
Mobile phases were prepared in hexane. 2-Hydroxy avonon. 3-Hydroxy avonon. d 1,10 -Bis-2-naphthol. e 10 mM bCD. f 10 mM 3-hydroxyl-propyl bCD. g 10 mM L-proline containing bCD. a
Conclusions
17
b
c
This journal is © The Royal Society of Chemistry 2014
18
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