Novel chiral stationary phase for the enantioseparation of racemic drugs 2

Chapter Effects of Spacer Length 5.1 Introduction In Chapter 4, we have studied the effects of substituents as chiral selectors and it appears that phenylcarbamate CSPs are suitable for separation of a wide range of racemic compounds. Apart from this, we understand that CSPs are usually prepared by immobilizing chiral molecules such as modified CDs onto the silica supports via appropriate alkyl spacers.1-3 It appears that the type and length of the spacers in the CSPs are also important factors in affecting the enantioselectivity in HPLC.4-11 It was reported that the enantioseparations of the CD CSPs are partially attributed to the spacer which kept the cavity of the chiral molecules away from the support surface.12 The length of the attachment of the tether connecting the chiral selector to the chromatographic support can have a dramatic influence in the chiral separation because the spacer makes the solutes’ access easier for the chiral discrimination to take place.13 In this chapter, we investigate the effects of spacer length of the phenylcarbamate CSP based on the basis of the work done in the previous chapter. In this work, three chemically bonded ether-linkage β-CD CSPs with different spacer lengths have been synthesized as illustrated in Figure 5.1. The three CSPs ETHE-3PC, ETHE-6PC and ETHE-11PC (with 3-, 6- and 11-carbon spacer respectively) were derived from the phenylcarbamoylated β-CD and immobilized onto the silica surface via a spacer arm with different lengths. The spacer length is varied in the number of methylene group (-CH2-) and the CSPs were packed into HPLC microbore column [∅ 2.1 x 150 mm] for HPLC analysis. 107 n( Si m O )( OR)7-n ( OR )14 m = no. of carbon Figure 5.1. CSPs with different spacer lengths. Surface coverage for the three CSPs was calculated based on the following formula.6 αexp = (%C x 106) / (1200Nc - %C.MW) 340 [µmol/m2] 5-1 where αexp is the surface concentration, %C is the percentage of carbon of the βCD CSP (from elemental analysis), MW is the relative molecular mass of the chiral ligand unit and Nc is the number of carbon in the total chiral ligand. Table 5.1. Elemental Analysis and Surface Concentration for the CSPs. CSP m Spacer length Elemental Analysis Carbon (%) αexp,ave* (µmol/m2) ETHE-3PC 3C 14.56 0.25 ETHE-6PC 6C 12.56 0.20 ETHE-11PC 11C 10.21 0.15 *The average surface coverage (αexp,ave) was calculated based on repeat experiments. 108 From Table 5.1, it is evident that ETHE-3PC has the highest surface loading, followed by ETHE-6PC and the lowest for ETHE-11PC. Shorter spacer arms such as the 3- and 6-carbon spacer might contribute lesser hindrance in the immobilization process leading to higher surface coverage. For the 11-carbon spacer, it is possible for the long and flexible chain to possess considerable degree of flexibility and rotational freedom that may reduce the immobilization efficiency and lead to a lower surface coverage. The efficiency of each CSP was determined under normal phase using biphenyl as standard. The efficiency was calculated to be ~40000 plates per meter for the three CSP columns. Chiral recognition abilities of the three CSPs were evaluated under normal and reversed phase. It was found that the length of the spacer contributes significantly to the retentivity, selectivity and resolution of the selected enantiomers. Based on the chiral recognition trends in resolving series of chiral compounds, the dependence of the enantioselectivity on the alkyl chain length was proposed. 5.2 Normal Phase Substituted (1-aryl) but-3-en-1-ols are useful synthetic precursors in organic syntheses. The alcohols were evaluated on the three CSPs under the same chromatographic conditions for comparison purposes as summarized in Table 5.2. It appears that the largest k’ values and highest enantioselectivity were observed on ETHE6PC for all the alcohols regardless of the position of substitution. 109 Table 5.2. Enantioseparations of substituted (1-aryl) but-3-en-1-ols under normal phase. S/n Compound 1. OH 2. OMe k’1 k’2 α Rs Spacer Length 0.54 0.80 0.49 0.60 0.87 0.56 1.11 1.21 0.98 0.27 0.40 0.20 3C6C11C- 1.31 1.87 1.21 1.44 2.04 1.37 1.09 1.10 0.88 0.43 0.46 0.40 3C6C11C- 0.67 0.91 0.67 0.89 1.21 0.88 1.33 1.34 1.21 0.78 0.92 0.73 3C6C11C- 0.60 0.91 0.60 0.88 1.19 0.79 1.31 1.45 1.11 0.95 1.02 0.67 3C6C11C- 0.78 1.08 0.75 1.29 1.69 1.12 1.56 1.68 1.40 1.17 1.38 1.07 3C6C11C- 0.58 0.83 0.58 0.73 1.02 0.64 1.24 1.25 1.11 0.60 0.70 0.36 3C6C11C- 0.98 1.43 1.04 2.31 3.26 2.48 2.36 2.48 2.07 3.89 4.44 3.75 3C6C11C- 0.87 1.34 0.71 1.29 1.98 0.81 1.47 1.48 1.14 1.83 2.10 0.43 3C6C11C- 0.56 0.73 0.46 0.67 0.80 0.54 1.18 1.18 1.08 0.36 0.40 0.32 3C6C11C- OH 3. F OH F 4. OH 5. Cl OH Cl 6. OH 7. Br OH 8. Ph OH 9. Me OH Conditions: Hexane/IPA=95/5 (v/v), flow rate at 0.1 ml/min. 110 Since most of the separated compounds showed the best chiral resolution results on ETHE-6PC, it is believed that the optimal spacer length for solute retention and selectivity was achieved on ETHE-6PC, which seems to be closed to methylene groups. It appears that solutes were allowed to interact more sufficiently on ETHE-6PC. A longer spacer arm on ETHE-6PC causes the CD selector further away from the silica surface and reduces the achiral H-bonding interaction between the solutes and the hydroxyl groups on the silica surface. The diminished participation of achiral molecular structures is helpful for the improvement of the enantioselectivities. In related studies done by the Chirosep group, a pentenyl moiety was determined to be the optimal spacer.14 Figure 5.2 gives the plot of separation factor (α) versus spacer arm length for the racemic samples. Separation Factor #1 #2 #3 #4 #5 #6 #7 #8 #9 Racemic Compound 3C 6C 11C Figure 5.2. Effects of spacer length on separation factor under normal phase. 111 Under normal phase, the highest enantioseparation abilities were afforded by the 6-carbon spacer CSP ETHE-6PC for selected flavanones in Table 5.3. It appears that CSP ETHE-6PC displays greater enantioseparation abilities than the other two CSPs for most of the test racemic compounds. Table 5.3. Enantioseparations of selected flavanones under normal phase. S/n Compound k’1 k’2 α Rs Spacer Length 1. 1.14 1.81 0.77 1.81 2.83 1.02 1.58 1.63 1.32 2.00 2.33 1.50 3C6C11C- 2.56 4.21 1.92 3.58 5.98 2.54 1.40 1.42 1.33 1.41 1.48 1.36 3C6C11C- 3.25 5.25 2.33 3.96 6.43 2.60 1.22 1.24 1.12 1.33 1.56 0.93 3C6C11C- 2.58 4.21 1.46 3.63 5.83 1.75 1.40 1.44 1.20 2.17 2.25 1.13 3C6C11C- 6.50 9.57 4.18 8.29 12.45 5.31 1.28 1.30 1.27 1.68 1.71 1.70 3C6C11C- 3.92 5.83 2.35 6.33 9.94 3.50 1.62 1.70 1.49 2.10 1.76 1.64 3C6C11C- Flavanone O O 2. 2’-Hydroxyflavanone HO O O 3. 6-Hydroxyflavanone O HO O 4. 4’-Methoxyflavanone OMe O O 5. 5-Methoxyflavanone O OMe 6. O 7-Methoxyflavanone MeO O O Conditions: Hexane/IPA=95/5 (v/v), flow rate at 0.1 ml/min. 112 3-Carbon spacer α = 1.58 6-Carbon spacer α = 1.63 11- Carbon spacer α = 1.32 Figure 5.3. Chromatograms of flavanone on the three columns under normal phase. Conditions: Hexane/IPA = 95/5 (v/v), flow rate at 0.1 ml/min. The chiral recognition ability of ETHE-11PC appears to be less satisfactory and afforded the poorest enantioseparations for all compounds in Table 5.2 and 5.3. The rotational freedom of the long spacer arm may have contributed to its poorer interaction with analytes. There exists a possibility that the mutual interactions between the CD chiral selectors would lead to a possible decrease of CSP-solute interactions (Figure 5.4). The poor solute retentivity on ETHE-11PC might also be attributed to its low surface loading. Often, stronger retention and enantioselectivity are observed for a CSP with higher surface loading. 113 Chiral Selector Analyte OH Si Chiral Selector OH Analyte Figure 5.4. Possible mutual interactions between chiral selectors. In summary, ETHE-6PC affords the highest solute retentivity and the best enantioselectivity for almost all the test racemic compounds under normal phase. Therefore, it is within reason to say the 6-carbon chain spacer appears to be the optimal spacer length for a phenylcarbamate CSP to achieve better enantioseparation under normal phase. 5.3 Reversed Phase The effects of spacer length were further investigated under reversed phase (Table 5.4). The dependence of the enantioselectivity on the spacer chain length under reversed phase was found to be similar to normal phase. In Table 5.4, ETHE-6PC affords the strongest retention and highest selectivity factor values as expected under reversed phase. 114 Table 5.4. Enantioselectivity of test racemic compounds under reversed phase. S/n 1. Compound Clenbuterol OH Cl H N C H k’1 k’2 α Rs 1.29 1.80 0.58 3.04 4.41 0.83 2.35 2.44 1.43 3.76 3.10 1.20 3C6C11C- 1.75 2.19 1.04 3.78 4.95 2.04 2.12 2.26 1.96 5.67 6.00 4.00 3C6C11C- 5.37 6.26 5.50 10.08 13.07 7.96 1.87 2.08 1.45 4.93 3.05 4.22 3C6C11C- 0.33 0.35 0.10 1.42 1.91 0.30 4.25 5.50 3.00 2.15 2.07 0.67 3C6C11C- 9.08 18.13 6.75 13.08 27.69 8.79 1.44 1.53 1.30 3.26 2.69 1.81 3C6C11C- 1.58 2.87 1.17 2.42 4.50 1.50 1.53 1.57 1.29 2.80 2.46 1.05 3C6C11C- 16.50 28.00 9.67 21.50 34.22 11.33 1.30 1.72 1.17 1.69 1.36 1.26 3C6C11C- 2.33 4.42 1.79 2.83 5.37 2.08 1.21 1.22 1.16 1.41 1.86 0.89 3C6C11C- Spacer Condition Length 1 H2N Cl 2. Atropine O H3C 3. N O OH Promethazine NMe2 N 1 1 S 4. Isoproterenol OH HO H N 1 HO 5. 4’-Hydroxyflavanone OH O 1 O 6. 5-Methoxyflavanone O 2 1 OMe O 2 Condition: at 1% TEAA pH 5.45/MeOH=60/40 (v/v); at water/MeOH=60/40 (v/v). UV detection at 254nm and flow rate at 0.2 ml/min. 115 7.1.5 Basic Chromatographic Parameters The concept of chromatography relies basically on the distribution of a compound between two phases, one of which (the mobile phase) is moving with respect to the other (the stationary phase). The essential part of a chromatographic system is the column that contains the stationary phase over which the mobile phase flows and where the separation of mixture into the individual components takes place. The sample is introduced by an injection device and the separated components are monitored by a suitable detection system. Ideally, the profile of a chromatographic band, as registered by the detector, should have a Gaussian distribution which gives a completely symmetrical peak. Figure 7.1. Retention and peak width. A chromatographic column may be characterized by its efficiency, which is a measure of column’s ability to transport a compound with little peak broadening. Here, biphenyl was selected as the standard in determining column efficiency under normal phase. Column efficiency is expressed as height equivalent to one theoretical plate, or plate height (H). H is readily calculated from using 142 N = 16 ( tR ) , H = L/N w 7.1-1 where tR is the retention time, w the baseline peak width, and L the column length. The retention of a compound on a column can be expressed by its retention time (tR) or capacity factor (k’), which is 7.1-2 k’ = (tR – to) / to When two components are involved, the separation factor α, is defined as α = (tR2 – to) / (tR1 – to) 7.1-3 It is important to recognize that separation factor (α) is a measure of relative peak separation and is constant under given analytical conditions (stationary and mobile phases, temperature etc). The resolution (RS) of two peaks in a chromatogram is dependent upon α and N. It shows how well the peaks are separated and can be easily evaluated according to RS = (tR2 – tR1) / (w1 + w2) 7.2 7.1-4 Preparation of Ether-linkage CSPs Partial-(6-O-propenylated)-β-CD 2a: Compound (40.00 g, 1.0 eq) was dissolved in 400 ml of DMF before allyl bromide (9.38 g, 2.2 eq) and sodium hydride (1.27 g, 1.5 eq) were added in. The mixture solution was stirred at 45°C for overnight. DMF was 143 removed and the residue was recrystalized from water. 2a was obtained as white solid (yield = 53.0 %). Melting Point > 300°C, decomposed; [α] 25 D +107.12° (c 0.01, H2O); IR (KBr, cm-1): 3379 (H-bonded O-H str overlapping with sp2 C-H str); 2927 (sp3 C-H str); 1640 (C=C str); 1417 (CH2 bend); 1157, 1080, 1028 (C-O str); 1H NMR (DMSO-d6) δ (ppm): 4.82 (H-1), 3.29 (H-2), 3.62 (H-3), 3.34 (H-4), 3.56 (H-5), 3.64 (H-6a,b), 5.72 (OH-2), 5.66 (OH-3), 4.46 (OH-6); 3.77 (triplet, 2H), 5.1-5.3 (multiplet, 2H), 5.74 (multiplet, 1H); 13C NMR (DMSO-d6) δ (ppm): 101.92 (C-1), 72.40 (C-2), 73.03 (C-3), 81.54 (C-4), 72.03 (C-5), 59.91 (C-6); 72.89 (-CH2-, 2H), 117.80 (=CH2, 2H), 134.63 (=CH, 1H); DEPT 135: 72.37, 72.52, 72.99, 81.50, 101.88, 134.63 (positive peaks); 59.87, 72.89, 117.80 (negative peaks); MALDI-TOF-MS m/z calcd for C45H74O35: 1175, found 1185 for [M]+; Elemental Analysis calcd for C45H74O35: C 45.99 %, H 6.30 %, found C 45.79 %, H 6.37 %. 5-Iodo-hex-1-ene: Triphenylphosphine (18.89 g, 1.2 eq), imidazole (4.94 g, 1.2 eq) and iodine (18.29 g, 1.2 eq) were added in order to 250 ml of dry dichloromethane. 5Hexen-1-ol (6.10 g, 1.0 eq) in 60 ml of dry dichloromethane was then added to the mixture and stirred at room temperature under nitrogen atmosphere for 3.0 hr. The mixture was filtered and the filtrate was concentrated and poured into a beaker of 400 ml of hexane. The top layer was removed and concentrated. Column chromatography was carried out for purification and a pale yellow liquid was obtained (yield = 66.3 %). IR (KBr, cm-1): 3074 (sp2 C-H str); 2929, 2854 (sp3 C-H str); 1640 (C=C str); 1427 (CH2 bend); 1215, 1172 (CH2-I bend); 724 (open chain CH2 bending); 1H NMR (CDCl3, TMS) δ (ppm): 1.50 (quintet, 2H); 1.84 (quintet, 2H); 2.07 (quartet, 2H); 3.21 (triplet, 2H); 4.98 144 (multiplet, 2H); 5.7-5.8 (multiplet, 1H); ESI-MS m/z calcd for C6H11I: 209, found 209 for [M]+ Partial-(6-O-hexenylated)-β-CD 2b: Compound (18.00 g, 1.0 eq) was dissolved in 400 ml of DMF before 5-iodo-hex-1-ene (7.33 g, 2.2 eq) and sodium hydride (0.57 g, 1.5 eq) were added in. The mixture solution was stirred at 45°C for overnight. DMF was removed and the residue was recrystalized from water. 2b was obtained as white solid (yield = 40.0 %). Melting Point > 300°C, decomposed; [α] 25 D +110.21° (c 0.01, H2O); IR (KBr, cm-1): 3389 (H-bonded O-H str overlapping with sp2 C-H str); 2927 (sp3 C-H str); 1638 (C=C str); 1414 (CH2 bend); 1155, 1080, 1029 (C-O str); 1H NMR (DMSO-d6) δ (ppm): 4.82 (H-1), 3.22 (H-2), 3.62 (H-3), 3.33 (H-4), 3.56 (H-5), 3.64 (H-6a,b), 5.72 (OH-2), 5.66 (OH-3), 4.44 (OH-6); 1.36 (quintet, 2H), 1.50 (quintet, 2H), 2.00 (quartet, 2H), 3.31 (triplet, 2H), 4.99 (multiplet, 2H), 5.7-5.8 (multiplet, 1H); 13C NMR (DMSO- d6) δ (ppm): 101.92 (C-1), 72.03 (C-2), 73.03 (C-3), 81.53 (C-4), 71.73 (C-5), 59.91 (C6); 24.46 (CH2, 2H), 28.70 (CH2, 2H), 32.81 (CH2, 2H), 72.40 (CH2-I, 2H), 114.87 (=CH2, 2H), 138.62 (=CH, 1H); MALDI-TOF-MS m/z calcd for C46H81O35: 1217, found 1238 for [M]+; Elemental Analysis calcd for C46H81O35: C 45.54 %, H 6.66 %, found C 44.96 %, H 6.92 %. 11-Iodo-undec-1-ene: Triphenylphosphine (18.88 g, 1.2 eq), imidazole (4.94 g, 1.2 eq) and iodine (18.29 g, 1.2 eq) were added in order to 250 ml of dry dichloromethane. 10-Undecenyl-1-ol (10.27 g, 1.0 eq) in 60 ml of dry dichloromethane was then added to the mixture and stirred at room temperature under nitrogen atmosphere 145 for 3.0 hr. The mixture was filtered and the filtrate was concentrated and poured into a beaker of 400 ml of hexane. The top layer was removed and concentrated. Column chromatography was carried out for purification and a pale yellow liquid was obtained (yield = 80.0 %). IR (KBr, cm-1): 3075 (sp2 C-H str); 2923, 2854 (sp3 C-H str); 1640 (C=C str); 1460 (CH2 bend); 1282, 1183 (CH2-I bend); 720 (open chain CH2 bending); H NMR (CDCl3, TMS) δ (ppm): 1.2-1.5 (singlet, 12H); 1.84 (quintet, 2H); 2.02 (quartet, 2H); 3.18 (triplet, 2H); 4.9-5.1 (multiplet, 2H); 5.7-5.9 (multiplet, 1H); ESI-MS m/z calcd for C11H21I: 280, found 279 for [M]+. Partial-(6-O-undecenylated)-β-CD 2c: Compound (20.00 g, 1.0 eq) was dissolved in 400 ml of DMF before 11-iodo-undec-1-ene (10.86 g, 2.0 eq) and sodium hydride (0.63 g, 1.5 eq) were added in. The mixture solution was stirred at 45°C for overnight. DMF was removed and the residue was recrystalized from water. 2c was obtained as white solid (yield = 52.0 %). Melting Point > 300°C, decomposed; [α] 25 D +116.67° (c 0.01, H2O); IR (KBr, cm-1): 3369 (H-bonded O-H str overlapping with sp2 C-H str); 2925, 2854 (sp3 C-H str); 1640 (C=C str); 1414 (CH2 bend); 1157, 1080, 1029 (C-O str); H NMR (DMSO-d6) δ (ppm): 4.82 (H-1), 3.28 (H-2), 3.63 (H-3), 3.34 (H-4), 3.56 (H-5), 3.64 (H-6a,b), 5.71 (OH-2), 5.66 (OH-3), 4.44 (OH-6); 1.2-1.4 (singlet, 12H), 1.62 (quintet, 2H), 1.99 (quartet, 2H), 3.31 (triplet, 2H), 4.93 (multiplet, 2H), 5.7-5.8 (multiplet, 1H); 13C NMR (DMSO-d6) δ (ppm): 101.92 (C-1), 72.40 (C-2), 73.03 (C-3), 81.53 (C-4), 72.02 (C-5), 59.91 (C-6); 28.0-33.0 (CH2, 16H), 72.89 (CH2-I, 2H), 114.61 (=CH2, 2H), 138.75 (=CH, 1H); MALDI-TOF-MS m/z calcd for C51H91O35: 1263, found 146 1324 for [M]+; Elemental Analysis calcd for C51H91O35: C 48.48 %, H 7.20 %, found C 48.51 %, H 7.66 %. 7.3 Synthesis of Perfunctionalized Carbamolylated β-CD Partial-(6-O-propenylated) perphenylcarbamolylated β-CD 3a: Compound 2a (3.50 g, 1.0 eq) was dissolved in 90 ml of dried pyridine and 10 ml of phenyl isocyanate was added in dropwise. The mixture was allowed to stir for 15.0 hr at 95 °C. Pyridine was removed and the remaining residue was extracted into ethyl acetate. The pre-concentrated ethyl acetate was added into a solvent mixture of hexane/ethyl acetate (6/1) dropwise with vigorous stirring for half an hour. Light yellowish precipitates were observed. The precipitates were filtered off and dissolved in ethyl acetate. The reprecipitation procedure was repeated for at least three times for complete purification. The purity of the product was checked using TLC. 3a (yield = 93.4 %). Melting point = 202.2-208.0°C; [α] 25 D +8.60° (c 0.01, CHCl3); IR (KBr, cm-1): 3393, 3312 (N-H str); 3059 (sp2 C-H str); 2960 (sp3 C-H str); 1734 (C=O str); 1602, 1444 (aromatic C=C str); 1539 (N-H bend); 1223, 1053 (C-O str); 1H NMR (CDCl3, TMS) δ (ppm): 7.2-8.2 (aromatic ring H overlapping with carbamate H); C NMR (CDCl3, TMS) δ (ppm): 120-140 (aromatic ring C), 152- 13 153 (carbamte C=O); MALDI-TOF-MS m/z calcd for C185H174N20O55: 3556, found 3556 for [M]+; Elemental Analysis calcd for C185H174N20O55: C 62.48 %, H 4.89 %, N 7.80 %, found C 60.43 %, H 3.48 %, N 8.10 %. 147 Partial-(6-O-hexenylated) perphenylcarbamolylated β-CD 3b: Same procedure as 3a except 2b was used to afford the corresponding 3b (yield = 77.3 %). Melting point = -1 172.7-177.2°C; [α] 25 D +14.89° (c 0.01, CHCl3); IR (KBr, cm ): 3394, 3307 (N-H str); 3138, 3059 (sp2 C-H str); 2956 (sp3 C-H str); 1716 (C=O str); 1602, 1442 (aromatic C=C str); 1539 (N-H bend); 1227, 1082, 1052 (C-O str); 1H NMR (CDCl3, TMS) δ (ppm): 7.28.2 (aromatic ring H overlapping with carbamate H); 13C NMR (CDCl3, TMS) δ (ppm): 120-140 (aromatic ring C), 152-153 (carbamte C=O); MALDI-TOF-MS m/z calcd for C189H182N20O55: 3612, found 3639 for [M]+; Elemental Analysis calcd for C189H182N20O55: C 62.84 %, H 5.03 %, N 7.75 %, found C 63.51 %, H 5.32 %, N 7.99 %. Partial-(6-O-undecenylated) perphenylcarbamolylated β-CD 3c: Same procedure as 3a except 2c was used to afford the corresponding 3c (yield = 80.0 %). Melting point = -1 180.1-185.3°C; [α] 25 D +22.78° (c 0.01, CHCl3); IR (KBr, cm ): 3393, 3303 (N-H str); 3059 (sp2 C-H str); 2926, 2854 (sp3 C-H str); 1737 (C=O str); 1600, 1444 (aromatic C=C str); 1541 (N-H bend); 1228, 1053 (C-O str); 1H NMR (CDCl3, TMS) δ (ppm): 7.2-8.2 (aromatic ring H overlapping with carbamate H); 13C NMR (CDCl3, TMS) δ (ppm): 120140 (aromatic ring C), 152-153 (carbamte C=O); MALDI-TOF-MS m/z calcd for C194H192N20O55: 3682, found 3692 for [M]+; Elemental Analysis calcd for C194H192N20O55: C 63.28 %, H 5.21 %, N 7.60 %, found C 63.46 %, H 5.24 %, N 8.04 %. Partial-(6-O-propenylated) per-1-naphthylcarbamolylated β-CD 3d: Same procedure as 3a except 1-naphthyl isocyanate was used to afford the corresponding 3d (yield = 90.0 -1 %). Melting point = 206.3-209.0°C; [α] 25 D +57.40° (c 0.01, CHCl3); IR (KBr, cm ): 148 3393, 3304 (N-H str); 3052 (sp2 C-H str); 2954 (sp3 C-H str); 1734 (C=O str); 1629, 1499 (aromatic C=C str); 1595, 1540 (N-H bend); 1213, 1103, 1037 (C-O str); 1H NMR (CDCl3, TMS) δ (ppm): 7.2-8.2 (aromatic ring H overlapping with carbamate H); 13 C NMR (CDCl3, TMS) δ (ppm): 120-140 (aromatic ring C), 152-153 (carbamte C=O); MALDI-TOF-MS m/z calcd for C265H214N20O55: 4557, found 4585 for [M]+; Elemental Analysis calcd for C265H214N20O55: C 69.84 %, H 4.70 %, N 6.14 %, found C 68.92 %, H 4.88 %, N 6.10 %. Partial-(6-O-propenylated) per-4-chlorophenylcarbamolylated β-CD 3e: Same procedure as 3a except 4-chlorophenyl isocyanate was used to afford the corresponding 3e (yield = 86.0 %). Melting point = 248.1-250.2°C; [α] 25 D +36.40° (c 0.01, CHCl3); H NMR (CDCl3, TMS) δ (ppm): 7.2-8.2 (aromatic ring H overlapping with carbamate H); C NMR (CDCl3, TMS) δ (ppm): 120-140 (aromatic ring C), 152-153 (carbamate C=O); 13 IR (KBr, cm-1): 3407, 3311 (N-H str); 3124, 3068 (sp2 C-H str); 2958 (sp3 C-H str); 1730 (C=O str); 1600, 1493 (aromatic C=C str); 1531 (N-H bend); 1224, 1091, 1041 (C-O str); 825 (para subst. oop); MALDI-TOF-MS m/z calcd for C185H154N20O55: 4245, found 4292 for [M]+; Elemental Analysis calcd for C185H154N20O55: C 52.35 %, H 3.63 %, N 6.60 %, Cl 16.70 %, found C 50.35 %, H 3.99 %, N 5.90 %, Cl 16.33 %. Partial-(6-O-propenylated) per-4-methoxyphenylcarbamolylated β-CD 3f: Same procedure as 3a except 4-methoxyphenyl isocyanate was used to afford the corresponding 3f (yield = 84.8 %). Melting point = 195.7-197.0°C; [α] 25 D -16.00° (c 0.01, CHCl3); IR (KBr, cm-1): 3390, 3309 (N-H str); 3070 (sp2 C-H str); 2954, 2835 (sp3 C-H 149 str); 1734 (C=O str); 1602, 1462 (aromatic C=C str); 1513 (N-H bend); 1227, 1174, 1030 (C-O str); 827 (para subst. oop); 1H NMR (CDCl3, TMS) δ (ppm): 7.2-8.2 (aromatic ring H overlapping with carbamate H); 13C NMR (CDCl3, TMS) δ (ppm): 120-140 (aromatic ring C), 152-153 (carbamate C=O); MALDI-TOF-MS m/z calcd for C205H214N20O55: 4156, found 4187 for [M]+; Elemental Analysis calcd for C205H214N20O55: C 59.24 %, H 5.15 %, N 6.74 %, found C 58.01 %, H 5.21 %, N 6.62 %. 7.4 Hydrosilylation and Immobilization 3.00 g of 3a-3f and 10 mg of tetrakis (triphenylphosphine) platinum were dissolved in ml of THF before another 10 ml of TES was added in. The solution was stirred at 80 °C for days. 2.50 g of 4a-4f was obtained as yellowish solid after removal of solvents. Moisture must be avoided throughout the reaction. 4a-4f was dissolved in 50 ml of toluene. 4.00 g of dry Kromasil silica gel was then added in. The mixture was refluxed for overnight. After removal of solvents, the residue was soxhlet extracted with acetone. The resultant CD CSP 5a-5f was obtained after removal of acetone. 5a [ETHE3PC] IR (KBr, cm-1): 1734 (C=O str); 1099 (C-O str); Elemental Analysis: C 14.56 %, H 1.77 %, N 1.42 %; Surface concentration: 0.25 mmol/m2g. 5b [ETHE-6PC] IR (KBr, cm-1): 1741 (C=O str); 1089 (C-O str); Elemental Analysis: C 12.56 %, H 1.47 %, N 1.29 %; Surface concentration: 0.20 mmol/m2g. 5c [ETHE-11PC] IR (KBr, cm-1): 1735 (C=O str); 1099 (C-O str); Elemental Analysis: C 10.21 %, H 1.01 %, N 0.48 %; Surface concentration: 0.15 mmol/m2g. 5d [ETHE-3NC] IR (KBr, cm-1): 1738 (C=O str); 1099 (C-O str); Elemental Analysis: C 16.61 %, H 1.39 %, N 2.10 %; Surface concentration: 150 0.20 mmol/m2g. 5e [ETHE-3pCPC] IR (KBr, cm-1): 1720 (C=O str); 1091 (C-O str); Elemental Analysis: C 12.56 %, H 1.36 %, N 1.02 %, Cl 3.17 %; Surface concentration: 0.22 mmol/m2g. 5f [ETHE-3pMPC] IR (KBr, cm-1): 1734 (C=O str); 1097 (C-O str); Elemental Analysis: C 13.74 %, H 1.39 %, N 0.92 %; Surface concentration: 0.21 mmol/m2g. 7.5 Synthesis of CSP 5g Hydride-modified silica gel 6: 5.00 g of Kromasil silica gel was placed in a RBF and a 100 ml of dioxane was then added in, followed by ml of aq. HCl solution (0.1M). The mixture was heated to about 70-80°C and 45 ml of 0.50M TES/dioxane solution was added slowly but continuously over a period of 15-20 min. The mixture was refluxed gently for 1~2 hr to yield the hydride-modified silica gel 6. The modified silica gel was filtered off from solvents and washed consecutively with 50ml portions of water/THF (20:80), THF and diethyl ether (twice with each solvent). The final product was dried at RT and then in a vacuum oven at 110°C for 6.0 hr or more. IR (KBr, cm-1): 2258 (Si−H str); 29Si CP-MAS NMR (TMS) δ (ppm): -74 (HSi*(OH)(OSi≡)2), -84 (HSi*(OSi≡)3). Immobilization of 2a: 1.00 g of 2a was dissolved in 40 ml of toluene. The mixture was stirred at 70°C for 1.0 hr after adding 0.1 ml of tert-butyl peroxide. 2.00 g of was then suspended in the solution. The mixture was heated at 100°C for 36 hr to afford CSP 5g. The CSP was filtered off and washed successively with toluene, methylene chloride and diethyl ether. The final CSP was soxhlet extracted and dried in vaccum at 100°C. 5g 151 [ETHE-3OH] IR (KBr, cm-1): 3437 (H-bonded O-H str overlapping with sp2 C-H str); 2927 (sp3 C-H str); 1637 (weak C=C str); 1080 (C-O str); Elemental Analysis: C 3.00 %, H 1.04 %; Surface concentration: 0.17 mmol/m2g. 7.6 Packing of CSPs 5a-5g was packed into HPLC stainless steel microbore column [∅ 2.1 x 150 mm]. 1.00 g of the CSP was suspended in ml of dioxane and ml of tetrachloromethane. The mixture was sonicated for 30 before the packing. A portion of 5a was packed into a HPLC analytical column [∅ 4.6 x 250 mm] and was labeled as 5a” [ETHE-3PC-L]. 3.50g of the CSP was suspended in 10 ml of dioxane and 20 ml of tetrachloromethane. The columns were conditioned with mobile phase before use. 152 Chapter Conclusions and Suggestions for Future Work 8.1 Conclusion Six novel β-CD bonded chiral stationary phases have been developed. The CSPs were synthesized with different functional groups and immobilized onto the silica gel via a stable ether-linkage with varied spacer lengths. The CSPs were evaluated under normal and reversed phase for their chiral recognition abilities on selected racemic compounds or drugs. Of which, CSP ETHE-3OH showed no enantioseparation towards selected chiral compounds under normal and reversed phase. Different perfunctionalized substituents on the CD hydroxyl groups can affect chiral recognition abilities of the CSPs. Of all the substituents on the CD rim, phenylcarbamate makes the corresponding CSP ETHE-3PC, the most effective in both normal and reversed phase separations. ETHE-3NC, ETHE-3pCPC and ETHE-3pMPC which were perfunctionalized with naphthylcarbamte, 4-chlorophenylcarbamte and 4methoxyphenylcarbamte respectively and shown to have significant enantioseparation preferences for selected chiral compounds or drugs. Phenylcarbamate CSP ETHE-3PC is commonly used for the enantioseparation of a wide range of structurally diverse racemic compounds. Naphthylcarbamate CSP ETHE-3NC showed good discriminating abilities towards amines and β-blockers. 4Methoxyphenylcarbamate CSP ETHE-3pMPC showed good enantioselectivity on imidazoles, piperazines, flavanones and particularly β-blockers. 4- 154 Chlorophenylcarbamate CSP ETHE-3pCPC on the other hand, appears to have good enantioselectivity on electron-rich analytes such as weak acids, carbonyls, alcohols, flavanones, ethers, epoxides, thiazoles and imidazoles. ETHE-3NC, ETHE-3pCPC and ETHE-3pMPC were able to afford satisfactory chiral discrimination under reversed phase but poor or even lost of chiral recognition under normal phase. In the study of the effects of spacer length, phenylcarbamate CSPs ETHE-3PC, ETHE-6PC and ETHE-11PC were prepared with different spacer lengths. The CSPs showed significant differences in solute retentivity and selectivity. It appears that a longer spacer arm does not necessarily contribute to higher enantioselectivity. From the study, there appears an optimal spacer length of a 6-carbon chain under normal and reversed phase. Study on the optimization of chromatographic conditions was also investigated. Optimization of enantioseparation was carried out by changing the mobile phase composition, buffer concentration and pH, selection of polar organic modifier and control of flow rate. Thermodynamic studies and the effects of surface loading were also investigated for better understanding and handling of chromatographic properties. In summary, the six CSPs proved to be effective chiral stationary phases towards a wide variety of selected racemates with excellent enantioseparation abilities and wide application ranges. 155 8.2 Suggested Future Works This project covers topics related to application of HPLC in the chiral separations of racemic drugs or compounds. Based on the work accomplished in this dissertation, future works with advantages should be concentrated on the following areas: 1) From analytical scale to preparative scale analysis. Due to the low yield of the allylated and alkenylated β-CD, ether-linkage CD CSPs are still struggling to perform on preparative scale analysis. Scaling up of synthesis and enantioseparations under semi-preparative or preparative scale are in the process. 2) Applications of CSPs in other chromatographic separation methods. Introduction of HPLC polar-organic mode separation is encouraging. More variety of buffers can be introduced. Better control of chromatographic properties and further thermodynamics studies can be carried out. The applications of the β-CD CSPs can also be extended to SFC, GC and CE. 3) Study on separation mechanisms. More precise study on chiral discrimination mechanisms can be predicted using molecular modeling. The technique provides clearer view on how chiral discrimination takes place. Endcapping of CSP can be carried out for better enantioseparation of basic compounds. 156 List of Publications 1. “Direct Resolution Of Flavanones Under Normal And Reversed Phase HPLC Using Novel Ether-linkage β-Cyclodextrin Bonded Chiral Stationary Phase” Mee-Yoon Lo, Siu-Choon Ng and Chi-Bun Ching. (will be submitted to Journal of Chromatography A) 2. “Effects Of Spacer Length For Ether-linkage β-CD CSPs On Selected Racemic Compounds Under Reversed Phase HPLC” Mee-Yoon Lo, Siu-Choon Ng and Chi-Bun Ching. (will be submitted to Journal of Chromatography A) 157 [...]... range 133 6.8.1 Theory The distribution coefficient (K) of solute between the stationary phase and the mobile phase in a chromatographic system is a function of the difference in free energy of the solute in two phases, ∆G° RT ln K = ∆G° = − (∆H ° − T∆S °) 6.8-1 where R is the gas constant, T is the absolute temperature, and ∆G° is the difference in the free energy of the solute in the two phases, ∆H °... such chiral separation is an enthalpy-driven separation in the whole tested temperature range 2. 5 k'1 k '2 3 2 1 Separation Factor Capacity Factor 4 2 1.5 1 0.5 0 0 5 8 12 16 20 24 28 32 36 40 5 8 12 16 20 24 28 32 36 40 Temperature Temperature Figure 6.11 Effect of temperature (°C) on k’ and α 135 1.6 ln k'1 0.8 ln k '2 1.4 0.7 ln Separation Factor 1 .2 ln k' 1 0.8 0.6 0.4 0 .2 0.6 0.5 0.4 0.3 0 .2 0.1... 4 2 0 #1 #2 #3 #4 #5 #6 Racemic Compound 3C 6C 11C Figure 5.5 Effects of spacer length on separation factor under reversed phase It shows that the effects of spacer length are similar under normal and reversed phase for the three CSPs Mobile phase conditions and chiral discriminating mechanisms have less influence on the alkyl chain length In both phases, the 11-carbon spacer CSP ETHE-11PC affords the. .. interaction of analytes with the CSP On the other hand, reducing the organic content in the mobile phase will result in significant peak-tailing and broaden peaks due to long retention time There exists an optimal mobile phase composition for each separation under normal and reversed phase. 4-6 6 .2. 1 Normal Phase In both normal and reversed phase, solute retentivity is influenced by the composition of the mobile... phases, ∆H ° and ∆S ° are the enthalpy and entropy of the transfer of solute from the mobile phase to the stationary phase respectively By substituting K with k’/Φ (K= k’/Φ is the phase ratio) gives RT ln (k’/Φ) = − (∆H ° − T∆S °) 6.8 -2 Rearrangement of equation 6.8 -2 gives the van’t Hoff equation:19 ln k’ = − ( ∆H ° ∆S ° )+( ) + lnΦ RT R 6.8-3 Equation 6.8-3 predicts that a plot of ln k’ vs 1/T will be... slope of − ( ∆H ° ∆S° ) and an intercept of [ ( ) + lnΦ] indicating that lnΦ is independent RT R on the temperature In addition, the separation factor (α= k 2/ k’1) is a measure of the 134 enantioselectivity and represents the difference in the free energy of interactions of the two enantiomers with the stationary phase: ln α = − ∆∆G° ∆∆H ° ∆∆S ° =−( )+( ) RT R RT 6.8-4 In this case, a plot of the ln... compete with the analytes for the CD cavity sites Hence, inclusion complexation of the analytes will not be favored and analytes tend to be eluted faster.7 This resulted in weaker retention and faster elution of the solutes 122 Table 6 .2 Separation of acebutolol under reversed phase TEAA/MeOH (v/v) k’1 k 2 α Rs A: 60/40 1.04 1.46 1.40 1.57 B: 70/30 1.96 2. 71 1.38 1.89 C: 80 /20 3. 62 4. 92 1.36 1.70 Condition:... ADP 22 0 Polarimeter Determination of pH was performed on a Metrohm 6 92 pH/Ion Meter 140 7.1.3 Packing of CSPs Kromasil silica gel was used for the immobilization of the CSPs MeOH was chosen as slurry solvent, the CSP was packed into HPLC analytical [∅ 4.6 x 25 0 mm] or microbore [∅ 2. 1 x 150 mm] stainless column using standard Alltech air compressor at a maximum pressure of 7500 psi for 20 ~30 min The. .. flow rate often gives better resolution because solutes are allowed to interact with the CSP sufficiently Control of the flow rate is therefore one of the easiest ways to optimize an enantioseparation though resolution of peaks within shorter retention time is always preferred Generally the flow rate of an analytical column is between 0.1 ml/min to 2. 0 ml/min depending on the dimension of the column... poorest chiral recognition abilities Based on the results, the 6carbon chain spacer on the ETHE-6PC appears to be the optimal spacer length under normal and reversed phase 5.4 Conclusion The present study has demonstrated that the chromatographic performances of the CD-based CSPs are largely influenced by spacer length; the separation factor α depends on the spacer arm length between the silica gel and the . attributed to the spacer which kept the cavity of the chiral molecules away from the support surface. 12 The length of the attachment of the tether connecting the chiral selector to the chromatographic. 3C- 1 28 .00 34 .22 1. 72 1.36 6C- 1 5-Methoxyflavanone O O OMe 9.67 11.33 1.17 1 .26 11C- 1 2. 33 2. 83 1 .21 1.41 3C- 2 4. 42 5.37 1 .22 1.86 6C- 2 1.79 2. 08 1.16 0.89 11C- 2 Condition:. 5.98 1. 42 1.48 6C- 2. O O OH 1. 92 2.54 1.33 1.36 11C- 3 .25 3.96 1 .22 1.33 3C- 5 .25 6.43 1 .24 1.56 6C- 3. 6-Hydroxyflavanone O O OH 2. 33 2. 60 1. 12 0.93 11C- 2. 58 3.63 1.40 2. 17