Synthesis and application of immobilized polysaccharide-based chiral stationary phases for enantioseparation by high-performance liquid chromatography

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Journal of Chromatography A, xxx (2014) xxx–xxx

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Review

Synthesis and application of immobilized polysaccharide-based chiral stationary phases for enantioseparation by high-performance liquid chromatography Jun Shen a , Tomoyuki Ikai b , Yoshio Okamoto a,c,∗ a Polymer Materials Research Center, Key Laboratory of Superlight Materials and Surface Technology, Ministry of Education, College of Materials Science and Chemical Engineering, Harbin Engineering University, Harbin 150001, PR China b Graduate School of Natural Science and Technology, Kanazawa University, Kakuma-machi, Kanazawa 920-1192, Japan c Graduate School of Engineering, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8603, Japan

a r t i c l e

i n f o

Article history: Received 16 April 2014 Received in revised form 10 June 2014 Accepted 11 June 2014 Available online xxx Keywords: Chiral HPLC Chiral stationary phase Immobilization Cellulose Amylose

a b s t r a c t Polysaccharide-based chiral stationary phases (CSPs) or chiral packing materials (CPMs) have been frequently employed for analyzing and separating various enantiomers by high-performance liquid chromatography (HPLC). The polysaccharide derivatives dissolved in a solvent are usually coated on silica gel to be used as CSPs. This means that some solvents, which can swell or dissolve the derivatives, cannot be used as the eluents in HPLC. In this review, various immobilization methods of the polysaccharide derivatives are described. The immobilization often reduces the chiral recognition ability compared to that of the corresponding coated-type CSPs. This problem can be overcome by the versatility of eluent selection for the immobilized CSPs. Enantioseparations of various racemates on the immobilized commercial columns using the non-standard eluents are briefly summarized. © 2014 Elsevier B.V. All rights reserved.

Contents 1. 2.

3.

4.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Immobilization methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Immobilization with diisocyanates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. Immobilization at a reducing terminal . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3. Immobilization by radical polymerization of vinyl groups . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.4. Immobilization by photoirradiation or thermal treatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.5. Immobilization using 6-azido and 6-epoxy groups . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.6. Immobilization by polycondensation of alkoxysilyl groups . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.7. Hybrid-type chiral packing materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Application of immobilized CPMs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. Comparison between coated-type and immobilized-type CPMs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. Compounds resolved on immobilized CPMs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Introduction Since Pasteur reported the first resolution (enantioseparation) of racemic tartrate in 1848 [1], the separation of enantiomers had

∗ Corresponding author at: #1E, 27 Miyahigashi-cho, Showa-ku, Nagoya 4660804, Japan. Tel.: +81 52 753 7292; fax: +81 52 753 7292. E-mail address: [email protected] (Y. Okamoto).

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been one of the most difficult separations because most of their physical and chemical properties are identical. However, in the past three decades, various chromatographic methods, such as gas (GC) and high-performance liquid chromatography (HPLC) with chiral stationary phases (CSPs), have significantly changed this situation [2–5], and recently, most chiral compounds can be resolved by these methods. These methods have been widely used for the determination of the enantiomeric excess (e.e.) of chiral compounds. Fig. 1 shows the percentage of e.e. determination methods reported

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respectively. Besides these coated-type CSPs, the immobilized-type CSPs (Chiralpak IA, IB, IC, ID, IE and IF) have recently become commercially available [17]. In this review, the synthetic methods and applications of immobilized polysaccharide-based chiral packing materials (CPMs) for HPLC will be mainly described. 2. Immobilization methods

Fig. 1. Distribution of e.e. determination methods from 1995 to 2012. The data were obtained from Tetrahedron Asymmetry published in 1995–2003 and JACS published in 2005–2012.

in the papers published in “Tetrahedron Asymmetry” from 1995 to 2003 and “Journal of the American Chemical Society (JACS)” from 2005 to 2012 [6–8]. About 300–350 papers were reported the e.e. determination in Tetrahedron Asymmetry and about 200–290 papers in JACS. Before 1970, the e.e. was mainly estimated based on the chiroptical properties, such as specific optical rotation ([˛]D ), and as shown in Fig. 1, this method has become obsolete. In the mid-1970s, chiral NMR shift reagents were developed for the e.e. determination [9]. However, because the NMR method can be applied to rather limited members of chiral compounds and its accuracy is not high compared to chromatographic methods, this method has been scarcely used. Currently, chiral HPLC is the most popular method, and more than 70% e.e. determinations have been performed by this method, which can also be used for preparative separations including many chiral pharmaceuticals. Recently, super critical fluid chromatography (SFC) with carbon dioxide as the main eluent has also attracted our attention as a new method without using organic solvents. The SFC usually uses the same CSPs as those for HPLC, and the separation results are rather similar to those in the normal phase HPLC with non-polar eluents. Among the many CSPs so far developed for HPLC, the CSPs derived from polysaccharides, such as cellulose and amylose, are the most prominent for the separations of various chiral compounds [8,10–16]. Fig. 2 shows the distributions of the e.e. determination methods, CSPs for chiral HPLC and types of polysaccharide-based CSPs reported in the Angewandte Chemie International Edition in 2012. In this year, the journal published about 2100 communication papers and at least 199 of them reported e.e. determinations. As well as the methods in 2012 in Fig. 1, HPLC, GC and SFC are the three main methods, and HPLC is the most important one. HPLC and SFC analyses were predominantly performed with the cellulose-based CSPs, Chiralcel OD and OJ, and amylose-based CSPs, Chiralpak AD and AS. The structures of these CSPs are shown as 1–4 in Fig. 3. OD (CSP 1a in Fig. 3) and AD (CSP 2a in Fig. 3) include several brand names, such as CellCoat (Kromasil), RegisCell (Regis), Eurocel 01 (Knauer), Lux Cellulose-1 (Phenomenex), Sepapak-1 (Sepaserve) and Chiral Cellulose-C (YMC), besides Chiralcel and Chiralpak (Daicel),

Polysaccharide derivatives can be immobilized on silica gel by various methods, such as (1) linkage with diisocyanates, (2) linkage at the reactive terminal, (3) radical polymerization of vinyl groups, (4) photo irradiation, (5) click reaction, and (6) polycondensation of the alkoxysilyl groups. To have high chiral recognition ability, the polysaccharide derivatives are usually required to have a regular helical structure. Therefore, it is well known that on the coated-type CSPs, particularly those derived from the cellulose benzoate derivatives, the coating conditions of the derivatives on silica gel significantly influence their chiral recognition ability [18–20]. The formation of a regular structure seems to be easier due to the intramolecular hydrogen bond within the polysaccharide chain for the carbamate derivatives compared to the esters [21]. The carbamate derivatives include most of the polysaccharide-based CSPs described above except for Chiralcel OJ consisting of cellulose 4-methylbenzoate. The change of chemical composition of a polysaccharide derivative by the introduction of reactive groups for immobilization often reduces the chiral recognition ability as a CSP compared with that of the original polysaccharide derivative. This means that the introduction of the minimum amount of the reactive groups is preferable if the immobilization can be efficiently attained. Furthermore, the immobilization of the polysaccharide derivatives often reduces their chiral recognition ability because this makes it difficult for the polysaccharide chain to take a regular structure. To obtain the immobilized CSPs with chiral recognition ability analogous to that of the coated-type phases, the degree of immobilization must be as low as possible [22–26]. This means that the introduction of a small amount of reactive groups on a polysaccharide chain is necessary. 2.1. Immobilization with diisocyanates Isocyanates, particularly phenyl isocyanates, are the agents with a suitable reactivity for the reaction with the hydroxyl groups of the polysaccharides, such as cellulose and amylose. Therefore, diisocyanates have been used to immobilize the polysaccharide derivatives coated on the silica gel surface. In 1987, this method was used to synthesize immobilized cellulose 3,5-dimethyl- and 3,5dichlorophenylcarbamates on 3-aminopropyl silica gel (A-silica), probably as the first immobilization of polysaccharide derivatives on silica gel (Fig. 4) [25]. In this method, 6-trityl-cellulose dissolved in chloroform was first coated on silica gel, then the cellulosecoated silica gel was produced by the treatment of 6-trityl cellulose with hydrochloric acid in methanol. The addition of a diisocyanate non-regioselectively links cellulose with amino propyl groups on the silica gel surface. The immobilization by the diisocyanate can also take place between the cellulose chains. These immobilized CPMs exhibited a slightly lower chiral recognition compared to the corresponding coated-type CPMs, particularly when a large amount of diisocyanate was used to attain a high immobilization. The formation of too many linkages disorders the regular structure of the polysaccharide chains, which must result in the lowering of the chiral recognition. Regioselective immobilizations at only the 6-position and at the 2- and 3-positions were also carried out using the cellulose 2,3-bis(phenylcarbamate) or 6-phenylcarbamate, respectively. The regioselective derivatives exhibited a slightly higher chiral

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Fig. 2. (A) Distribution of e.e. determination methods reported in Angew. Chem. Int. Ed. in 2012. The numbers in parentheses show the number of papers. (B) CSPs for HPLC and SFC. (C) Polysaccharide-based CSPs. OD, AD, OJ and AS are coated-type CSPs and IA, IB, IC, ID and IE are immobilized-type CSPs. The structures of these CSPs are shown in Fig. 3. OD (CSP 1a in Fig. 3) and AD (CSP 2a in Fig. 3) include several brand names, besides Chiralcel and Chiralpak of Daicel, respectively.

Fig. 3. CSPs used in Daicel chiral columns. CSPs 1a, 2a and 2e have been commercialized with different brand names by several other companies.

recognition to many racemates than the above non-regioselectively immobilized CPMs [27]. Chen et al. reported the immobilized CSPs derived from other cellulose derivatives using diisocyanates [28,29]. They also used this method for the preparation of the CSPs for capillary chromatography [30,31]. This immobilization method was applied to the organic polymer-based support in place of the silica gel [32]. 2.2. Immobilization at a reducing terminal As already explained, in order to obtain the immobilized CPMs with a high chiral recognition, the polysaccharide derivatives must be immobilized with a low number of chemical bonds. The

immobilization at a terminal of a polysaccharide chain seems to be very attractive. As shown in Fig. 5, an amylose chain can be attached to the silica gel at the reducing terminal, although the process is not straightforward [33]. First, an amylose oligomer, maltopentaose, was oxidized at the reducing end to a lactone. This oligomer was then extended to a polymer using ␣-d-glucose 1-phosphate dipotassium and potato phosphorylase as the catalyst [34]. The amylose with the lactone end was allowed to react with A-silica, followed by the reaction with an excess 3,5-dimethylphenyl isocyanate to convert the hydroxy groups to carbamate residues. The obtained CPM exhibited a chiral recognition analogous to that of the coated-type CSP. However, this method can be applied only to amylose, therefore, immobilized cellulose-based CPMs cannot be similarly synthesized.

Fig. 4. Immobilization of cellulose derivatives using diisocyanate.

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Fig. 5. Immobilization of amylose derivatives at an activated reducing terminal.

Fig. 6. Structures of cellulose derivatives 5 and 6 bearing vinyl groups.

2.3. Immobilization by radical polymerization of vinyl groups Immobilization has been examined by the radical polymerization of several cellulose and amylose derivatives bearing vinyl groups. In 1993, Kimata et al. synthesized cellulose 4-vinylbenzoate 5 as shown in Fig. 6 and coated it on silica gel having acryloyl groups [35]. The 5-coated silica gel was suspended in a solvent and heated with a radical initiator. The obtained immobilized benzoate could be used with various solvents, but only a poor chiral recognition was attained probably due to too many cross-linkages, which disturb the formation of the regular structure of the cellulose chains. An analogous immobilization by radical polymerization was also performed using 6 shown in Fig. 6 bearing 10-undecenoyl (4–12%) and 3,5-dimethylphenylcarbamate (88–96%) and allyl silica gel [36–41]. The chiral recognition abilities of the resulting CPMs seem to be not as good as that of the coated-type CSP. This method was also applied to other phenylcarbamate derivatives of cellulose. In addition, the immobilization of the phenylcarbamate derivatives of amylose and chitosan were examined by this method [39–41]. As shown in Fig. 7, the immobilization of cellulose 3,5-dimethylphenylcarbamate derivatives with more reactive vinyl groups like 4-vinylphenylcarbamate 7 and 2-methacryloyloxyethylcarbamate 8 at 6-position was carried out in the presence of inert hydrocarbon monomers such as styrene and 2,3-dimethyl-1,3-butadiene [22,23,42]. The addition of styrene significantly improved the immobilization efficiency of 7 coated on both A-silica and M-silica (Fig. 8), and the immobilization efficiency on the M-silica was only slightly higher than that on the A-silica, suggesting that the vinyl groups on the M-silica are not the main factor for the immobilization, but the immobilization mainly occurs through the formation of a network between the cellulose derivatives. Table 1 shows the immobilization results of

Fig. 7. Structures of cellulose derivatives bearing 4-vinylphenylcarbamate (7) and 2-methacryloyloxyethylcarbamate (8).

7 in the absence and presence of styrene. The immobilization was carried out by heating 7 coated on A-silica with a radical initiator, ␣,␣ -azobisisobutyronitrile (AIBN) in hexane at 60 ◦ C. While in the absence of styrene, only 50% of 7 coated on the silica gel was immobilized to give the CPM-1, the addition of 10 wt% styrene of 7 enabled the almost quantitative immobilization of 7, yielding CPM-3. The recognition abilities of five immobilized CPMs (1–5) were evaluated for racemates 9–18 in Fig. 9 using a hexane/2propanol (90/10) mixture as the eluent. Good enantioseparation seems to occur on the CPMs immobilized with 5–10 wt% styrene. The cellulose derivative 8a coated on the A-silica was also immobilized by radical polymerization using 10 wt% of 2,3dimethyl-1,3-butadiene (DMBD) as a monomer to give CSP-6 as shown in Table 2 [43,44]. The immobilization efficiency (88%) was lower than that (99%) for CPM-3. The chiral recognition of the immobilized 8a seems to be slightly better than that of CPM-3 in Table 1. However, these chiral recognitions by CPM-3 and CPM-6 are still not comparable to that of the coated-type cellulose 3,5dimethylphenylcarbamate (1a) for several racemates. One of the reasons for the lower ability of the immobilized CPMs must be ascribed to the rather high content of vinyl groups in these derivatives, which may disturb the regular structure of the cellulose chain. Therefore, the derivative 8b with a lower methacryloyl content (4%) compared to 8a was synthesized and immobilized on the M-silica with DMBD. Although the immobilization efficiency decreased as expected, the obtained CPM-7 exhibited a slightly higher recognition. The chiral recognition of CPM-7 seems rather similar to that of the Chiralpak IB consisting of the immobilized CSP-1a. Compared to the coated-type 1a, all the immobilized-type 1a so far

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Fig. 8. Immobilization of polysaccharide derivatives onto (A) A-silica (see Fig. 4) and (B) M-silica by means of copolymerization with vinyl monomer. (Reproduced with permission from Ref. [26].)

Table 1 Influence of styrene content on immobilization efficiency of 7 and separation factors (␣) for racemates 9–18 on the CPMs-1–5 [23].

9 10 11 12 13 14 15 16 17 18

CPMs

CPM-1

CPM-2

CPM-3

CPM-4

CPM-5

Styrene content to 7 Immobilized fraction

0 wt% 50% 1.26 (−) 1.45 (+) ∼1 (−) ∼1 (+) 2.74 (−) 1.25 (+) 1.23 (−) ∼1 (+) 1.92 (−) 1.40 (+)

5 wt% 86% 1.22 (−) 1.45 (+) 1.33 (−) ∼1 (+) 2.57 (−) 1.34 (+) 1.22 (−) 1.17 (+) 2.18 (−) 1.42 (+)

10 wt% 99% 1.32 (−) 1.68 (+) ∼1 (+) 1.12 (+) 3.20 (−) 1.18 (+) 1.13 (−) 1.32 (+) 1.96 (−) ∼1 (+)

30 wt% >99% 1.31 (−) 1.60 (+) 1.17 (+) ca.1 (+) 2.76 (−) 1.16 (+) 1.08 (−) ca1 (+) 1.81 (−) ca1 (+)

50 wt% >99% 1.31 (−) 1.53 (+) 1.23 (+) ∼1 (+) 2.63 (−) 1.14 (+) ∼1 (−) ∼1 (+) 1.73 (−) ∼1 (+)

The signs in parentheses represent the optical rotation of the first-eluted enantiomer. Column: 25 cm × 0.20 cm (i.d.). Eluent: hexane–2-propanol (90:10). Flow rate: 0.1 ml/min. Silica gel: A-Silica. [Vinyl group]/[AIBN] = 50. Solvent for polymerization; hexane.

synthesized seems to show lower ability, although this depends on the racemates. In the cellulose derivatives 7 and 8, vinyl groups were regioselectively introduced at the 6-position of the glucose unit, since this position is less important for chiral recognition and is more suitable to produce a higher reactivity of the vinyl groups. Actually, these derivatives were smoothly immobilized on the silica gels. However, their synthesis is more laborious compared to the non-regioselective synthesis of the carbamate derivatives having vinyl groups at any of the 2-, 3- or 6-positions. Non-regioselective

derivatives 19–21 can be synthesized in a one-pot process as described in Fig. 10 [23,45]. For this process, in order to attain a uniform distribution of the side groups on the cellulose chain, the cellulose must be first dissolved in a mixture of N,N-dimethylacetamide (DMA) and LiCl. A part of the 3,5dimethylphenyl isocyanate is then allowed to react with impurities like water as well as with cellulose, followed by the addition of the desired amount of olefinic reagents used for the introduction of the vinyl groups, such as R4 , R5 and R6 . After coating on the MA-silica shown in Fig. 10, these derivatives were immobilized

Fig. 9. Structures of racemates 9–18.

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Table 2 Influence of the contents of vinyl group on immobilization of 8 and chiral recognitions to racemates 9–18 on the CPMs-6–8 and Chiralpak IB [43,44].a CPMs

CPM-6b

CPM-7c

CPM-8

Chiralpak IBd

Cellulose derivatives

8a (R2 /R4 = 90/10)

8b (R2 /R4 = 96/4)

Coated 1a

Immobilized 1a

Immobilized fraction

88%

78%

␣

k1 

␣

k1 

␣

k1 

␣

1.30 (−) 1.71 (+) 1.20 (−) 1.15 (+) 3.80 (−) 1.27 (+) 1.18 (−) 1.20 (+) 2.16 (−) ∼1(+)

1.23 (−) 0.81 (+) 0.75 (−) 1.59 (+) 2.05 (−) 2.56 (+) 1.46 (−) 0.42 (+) 1.82 (−) 1.51 (+)

1.27 1.64 1.49 1.17 3.83 1.34 1.23 1.13 2.42 1.40

0.76 (−) 0.75 (+) 0.48 (−) 1.06 (+) 1.15 (−) 1.50 (+) 0.96 (−) 0.42 (+) 1.38 (−) 0.61 (+)

1.17 1.31 1.96 1.12 2.40 1.50 1.34 ∼1 2.77 1.99

1.00 (−) 0.96 (+) 0.55 (−) 1.12 (+) 1.48 (−) 2.00 (+) 1.13 (−) 3.15 (+) 1.54 (−) 0.86 (+)

1.14 1.22 1.77 1.22 2.72 1.33 1.26 ∼1 2.42 1.89

9 10 11 12 13 14 15 16 17 18

–

–

a The signs in parentheses represent the optical rotation of the first-eluted enantiomer. Column: 25 cm × 0.20 cm (i.d.). Eluent: hexane–2-propanol (90:10). Flow rate: 0.1 ml/min. b Silica gel: A-Silica. Vinyl monomer: 2,3-dimethyl-1,3-butadiene (10 wt%). [Vinyl group]/[AIBN] = 30. Solvent for polymerization: toluene. c Silica gel: M-Silica. Vinyl monomer: 2,3-dimethyl-1,3-butadiene (30 wt%). [Vinyl group]/[AIBN] = 30. Solvent for polymerization: toluene. d Column: 25 cm × 0.46 cm (i.d.). Flow rate: 0.5 ml/min.

Fig. 10. Synthesis of cellulose derivatives 19–21 bearing vinyl groups at 2-, 3- or 6-positions.

by the radical copolymerization with 1,5-hexadiene by the AIBN at 80 ◦ C. The results of the immobilization are summarized in Table 3. Although a rather large amount (45 wt% of cellulose derivative) of 1,5-hexadiene was employed, the immobilization efficiencies,

particularly for the derivative 21, were low compared to the previous immobilizations with styrene and 2,3-dimethyl-1,3-butadiene. This must be associated with the poor reactivity of the nonconjugated olefinic group of 21. While the derivative 19 is linked via

Table 3 Immobilization efficiencies of cellulose derivatives 19–21 and chiral recognitions for racemates 9–18 on the CPMs-9–11 [45]. CPMs

CPM-9

CPM-10

CPM-11

Cellulose derivatives

19

20

21

Immobilized fraction

87%

9 10 11 12 13 14 15 16 17 18

78%

33%

k1 

␣

k1 

␣

k1 

␣

1.34 (−) 0.97 (+) 0.82 (−) 1.89 (+) 2.05 (−) 2.73 (+) 1.65 (−) 0.54 (+) 2.30 (−) 1.35 (+)

1.23 1.52 1.53 ∼1 2.75 1.37 1.22 1.10 2.24 1.39

0.95 (−) 0.77 (+) 0.59 (−) 1.18 (+) 1.51 (−) 1.92 (+) 1.12 (−) 0.38 (+) 1.54 (−) 0.96 (+)

1.24 1.34 1.66 1.23 3.72 1.43 1.27 ∼1 2.61 1.96

0.30 (−) 0.23 (+) 0.18 (−) 0.40 (+) 0.58 (−) 0.63 (+) 0.37 (−) 0.14 (+) 0.58 (−) 0.34 (+)

1.17 1.47 1.75 1.06 2.30 1.33 1.21 ∼1 2.13 1.52

Silica gel: MA-Silica in Fig. 10. Vinyl monomer: 1,5-hexadiene (45 wt%). [Vinyl group]/[AIBN] = 30. Solvent for polymerization: toluene. The signs in parentheses represent the optical rotation of the first-eluted enantiomer. Column: 25 cm × 0.20 cm (i.d.). Eluent: hexane–2-propanol (90:10). Flow rate: 0.1 ml/min.

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Fig. 11. Immobilization of the polysaccharide derivatives bearing azido (22) and hydroxy (23) groups onto A-silica (A) and G-silica (B), respectively.

solvents, like chloroform, which cannot be used for the coatedtype CSPs. They also mentioned that an analogous immobilization is also possible by the thermal treatment of the coated cellulose derivatives [47]. 2.5. Immobilization using 6-azido and 6-epoxy groups

Fig. 12. Carbamate derivatives of cellulose (24) and amylose (25) bearing trialkoxysilyl groups.

a carbamate group to a longer side chain methacrylate residue, 20 is directly linked via an ester bond to the methacryloyl group. This structural difference must influence the immobilization efficiency of these derivatives. Chiral recognitions of CPMs-9 and -10 were slightly different depending on the racemates. Some compounds were better resolved on CPM-9 and other compounds on CPM-10. The lower immobilized 21 content on CPM-11 resulted in a slightly lower recognition, probably because a non-enantioselective interaction between the racemates and silica surface more frequently occurred. The immobilization of the amylose derivatives with a vinyl group was also carried out in the same way [43,45]. 2.4. Immobilization by photoirradiation or thermal treatment Francotte and Huynh reported that cellulose chlorophenylcarbamate derivatives, such as the 3,5-dichloro- and 3,4dichlorophenylcarbamates, coated on silica gel are immobilized by UV light irradiation [46]. Although the immobilization mechanism is not clear, the immobilized CPMs can be used with prohibited

The immobilization of polysaccharide derivatives was also examined using the 6-azido-2,3-phenylcarbamate derivative 22 and A-silica shown in Fig. 11 [48,49]. The silica gel (G-silica) containing epoxy groups has been used for the immobilization of a cellulose derivative 23 having a free hydroxyl group at the 6-position, which can be readily synthesized from the 6-trityl cellulose shown in Fig. 4 [50]. A cationic BF3 ·OEt2 catalyst was added for opening the epoxy groups. Based on these procedures, only the silica surface can attach to the polysaccharide derivatives, thus their contents are rather low compared to the other methods, which allow the formation of a linkage between the polysaccharide chains. Therefore, these methods may be more suitable for introducing the polysaccharide derivatives on the capillary columns [49,50]. 2.6. Immobilization by polycondensation of alkoxysilyl groups The alkoxysilyl groups, such as 3-triethoxysilylpropyl, are known to readily polymerize by acid and base catalysis to form polysiloxanes. Actually, this reaction has been used for the production of silica gel. This high reactivity of the alkoxysilyl group can be utilized for immobilizing the polysaccharide derivatives. In 2003, Chen et al. tried to introduce the 3-triethoxysilylpropylcarbamate group using the 3-triethoxysilylpropyl isocyanate on cellulose [51]. However, they could introduce this group to cellulose only as an undetectable amount based on the NMR analysis. Therefore, they were unable to effectively use this group for the immobilization.

Fig. 13. Immobilization of polysaccharide derivatives via intermolecular polycondensation of triethoxysilyl groups. (Reproduced with permission from Ref. [26].)

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Table 4 Immobilization of 24a–d and separation factors (␣) for racemates 9–18 on the immobilized CPMs-12–15 and the coated-type CPM-8 [24]. CPMs Cellulose derivatives Immobilized fraction

CPM-12 24a (R2 /R8 = 96/4) 99%

CPM-13 24b (R2 /R8 = 97/3) 97%

CPM-14 24c (R2 /R8 = 98/2) 89%

CPM-15 24d (R2 /R8 = 99/1) 72%

CPM-8 1a (R2 /R8 = 100/0) –

9 10 11 12 13 14 15 16 17 18

1.27 (−) 1.52 (+) 1.21 (−) 1.17 (+) 3.66 (−) 1.23 (+) 1.13 (−) ∼1 (+) 2.01 (−) 1.22 (+)

1.31 (−) 1.56 (+) 1.30 (−) 1.18 (+) 4.36 (−) 1.31 (+) 1.19 (−) ∼1 (+) 2.29 (−) 1.48 (+)

1.23 (−) 1.53 (+) 1.51 (−) 1.15 (+) 3.52 (−) 1.35 (+) 1.21 (−) ∼1 (+) 2.35 (−) 1.65 (+)

1.27 (−) 1.49 (+) 1.59 (−) 1.19 (+) 3.70 (−) 1.35 (+) 1.23 (−) ∼1 (+) 2.47 (−) 1.70 (+)

1.17 (−) 1.31 (+) 1.96 (−) 1.12 (+) 2.40 (−) 1.50 (+) 1.34 (−) ∼1 (+) 2.77 (−) 1.99 (+)

The signs in parentheses represent the optical rotation of the first-eluted enantiomer. Eluent: hexane–2-propanol (90:10). Column: 25 cm × 0.20 cm (i.d.). Flow rate: 0.1 ml/min.

Table 5 Immobilization of 25a–d and separation factors (␣) for racemates 9–18 on the immobilized CPMs-16–19 and the coated-type CPM-20 [24]. CPMs Amylose derivatives Immobilized fraction

CPM-16 25a (R2 /R8 = 97/3) >99%

CPM-17 25b (R2 /R8 = 98/2) 99%

CPM-18 25c (R2 /R8 = 99/1) 86%

CPM-19 25d (R2 /R8 = >99/<1) 28%

CPM-20 2a (R2 /R8 = 100/0) –

9 10 11 12 13 14 15 16 17 18

∼1 (−) 1.37 (+) 2.37 (+) 1.88 (+) 2.18 (−) 1.09 (−) 1.14 (+) 1.0 1.0 3.51 (+)

∼1 (−) 1.43 (+) 2.55 (+) 2.00 (+) 2.24 (−) 1.14 (−) 1.08 (+) 1.0 1.0 3.08 (+)

∼1 (−) 1.44 (+) 2.83 (+) 2.11 (+) 2.27 (−) 1.18 (−) 1.08 (+) 1.0 1.0 3.49 (+)

∼1 (−) 1.21 (+) 3.08 (+) 2.17 (+) 2.27 (−) 1.18 (−) 1.0 1.0 1.0 4.61 (+)

∼1 (−) 1.60 (+) 3.33 (+) 2.02 (+) 2.21 (−) 1.31 (−) ∼1(+) 1.0 1.36 (+) 2.54 (+)

The signs in parentheses represent the optical rotation of the first-eluted enantiomer. Eluent: hexane–2-propanol (90: 10). Column: 25 cm × 0.20 cm (i.d.). Flow rate: 0.1 ml/min.

The above reaction was carried out under different conditions and the introduction of controlled amounts of the 3-triethoxypropylcabamate (R8 ) groups to cellulose was attained (Fig. 12) [24]. By this method, the cellulose derivatives 24 with 1–4% of R8 contents were synthesized. After being coated on the silica gel, the derivatives were immobilized by heating with a mixture of ethanol/water/(CH3 )3 SiCl for 10 min at 110 ◦ C (Fig. 13). The immobilization results are summarized together with the recognition data of the obtained CPMs-12–15 (Table 4). Even 24b with a 3% R8 enabled an almost quantitative immobilization, indicating the much higher activity of R8 for immobilization than the other groups discussed above. The chiral recognition abilities of CPMs-13–15 are very close to each other, and that of CPM-12 with a 4% R8 seems to be slightly lower than the others, suggesting that the introduction of a few percent R8 may be suitable from the viewpoints of the immobilization efficiency and chiral recognition. Compared to the coated-type CSP-8, several racemates are better resolved on the immobilized CSPs and several racemates on CSP-8. The immobilization even by a very small amount of R8 may still change the structure of the cellulose derivative 1a. The amylose derivatives 25a–d were synthesized in the same way and immobilized on silica gel (Table 5) [24]. The amylose derivatives seems to be more efficiently immobilized on silica gel than the cellulose derivatives with the same R8 contents, and with even a 2% R8 , an almost complete immobilization was realized. The chiral recognitions of CPMs-16–18 with 1–3% R8 are very similar to each other, but slightly different from that of the CPM-20 coated with pure 2a. Racemates 10, 11, 14 and 17 were better resolved on CPM-20, while racemates 15 and 18 were better on the immobilized CPMs. Again, the immobilization seems to change the structure of the amylose derivative.

As described above, the chiral recognitions of these immobilized CPMs were slightly low or changed compared to the coated-type CSPs. However, this disadvantage is often overcome using many prohibited eluents, which cannot be used on the coated-type CSPs, as will be discussed later. 2.7. Hybrid-type chiral packing materials The polysaccharide derivatives 24 and 25 can also be utilized for the preparation of the spherical hybrid-type CPM, which consists of a nanocomposite of silica gel and the polysaccharide derivative formed by a sol–gel reaction in an aqueous surfactant solution [52]. The CPM is a totally organic–inorganic hybrid material, and much higher contents of the immobilized polysaccharide derivatives compared to the CPMs prepared via coating process are possible by this method. The high content of the polysaccharide derivatives is preferable for the preparative separation due to the higher loading capacity of the CPMs. This hybrid CPM has been used for the resolution of ␤-blockers [53]. 3. Application of immobilized CPMs As already mentioned, compared to the coated-type CPMs, the immobilized CPMs have several merits including (1) higher stability of CSPs, (2) wider eluent selection for analytical and preparative separation, and (3) easy recovery of the chiral recognition ability of the CSPs with some treatment after partial losing of chiral recognition ability, etc. In 2004, the first immobilized CPMs were commercialized as Chiralpak IA and Chiralpak IB from Daicel. The structures of these CSPs including four other Chiralpak, IC, ID, IE and IF, are shown in Fig. 3. Among these, the CSPs of Chiralpak

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Fig. 14. Compounds resolved on Chiralpak IA using various eluents.

IA, IB and IF had already been commercialized as the coated-type columns Chiralpak AD, Chiralcel OD and Chiralpak AZ, respectively, and the other three, Chiralpak IC, ID and IE, are available only as the immobilized-type, mainly due to the high solubility of these polysaccharide derivatives. While the high chiral recognition ability of cellulose 3,5-dichlorophenylcarbamate (1b) was reported in 1986 [54], it had not been commercialized because of its high solubility in hexane containing alcohols.

3.1. Comparison between coated-type and immobilized-type CPMs As already discussed, the immobilized-type CPMs are more or less different from the coated-type CPMs in chiral recognition, and often show slightly lower ability, because the formation of a regular structure of the polysaccharide chains becomes difficult by the incorporation of the different side groups for the

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Fig. 15. Compounds resolved on Chiralpak IB, IC and ID.

immobilization. Using 48 racemates and the eluents suitable for the coated-type CPMs, Andersson et al. compared the recognition abilities of the immobilized-type CPMs, Chiralpak IA and Chiralpak IB, with the corresponding coated-type CPMs, Chiralpak AD and Chiralcel OD, respectively [55], and confirmed that coated-type CPMs often exhibit better recognitions. However, the weak point of the immobilized-type CPMs may be overcome by suitable selection of the prohibited eluents, for examples, chloroform (CHCl3 ), dichloromethane (CH2 Cl2 ), tetrahydrofuran (THF), methyl t-butyl ether (MtBE) and ethyl acetate (EA) as described below. 3.2. Compounds resolved on immobilized CPMs Fig. 14 shows examples of racemates 26–42 resolved on the immobilized amylose 3,5-dimethylphenylcabamate, Chiralpak IA,

and the eluents used for their resolution [55–70]. Most of the eluents cannot be used for the corresponding coated-type column Chiralpak AD and often provided better separation compared to the standard eluents for the coated-type CPMs. Compounds 43–58 resolved on Chiralpak IB, IC and ID are shown in Fig. 15 [17,71–76]. Chiralpak IB in most cases exhibits a lower chiral recognition than its coated-type column, Chiralcel OD, when the typical hexane-alcohol eluents were used [72,73,77]. Again, with non-standard eluents, this problem has been improved. Chiralpak IC and ID with an electron withdrawing substituent expand the possibility of enantioseparation [17]. The high stability of the immobilized CPMs allows them to be used for the direct analysis of reaction mixtures carried out in the prohibited solvents like dichloromethane [78,79].

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