**2. The development of cellulose-based CSPs**

The cellulose-based CSPs generally are of two types: the coated and the bonded. The coated cellulose-based CSPs consisting of the low-molecular-weight cellulose benzoate or phenyl carbamate showed higher chiral recognition than the covalently bonded CSPs for most racemates. The major reason was considered to be an optimal secondary and supermolecular structure for the chiral recognition mechanism of polysaccharide derivatives under coated conditions [1,3]. However, the coated CSPs can only be used with a limited range of solvents as mobile phases such as alkanes, alcohols, acetonitrile, or aqueous solvents including alcohols or acetonitrile because CSPs may dissolve in 'strong' solvents such as tetrahydrofuran (THF) and chloroform (CHCl3). Such a dissolution would damage or destroy the CSPs. This limited the application range of the coated CSPs on separation and preparation of chiral compounds, because the solubility of the sample in the mobile phase is very important to increase the amount of racemates loaded on CSPs, especially on a preparative large-scale separation [7].

The bonded CSPs were prepared by covalently bonding cellulose derivates to silica gel. They can be applied to a wider range of resolving conditions than the coated type. The fixation can affect the conformation of cellulose derivates and make it difficult to obtain optimal supermolecular structure. This results in lower chiral recognition ability of the bonded-type CSPs. However, the fixation improves versatility in the solvent selection, and allows the use of some solvents that cannot usually be applied on the coated CSPs as mobile phases or sample dissolving reagents [8].

The commercial cellulose-based CSPs including the coated and the bonded CSPs currently in use are summarized in Table 1. As can be seen, there are only two columns (Chiralpak IB and Chiralpak IC) prepared from cellulose derivatives by bonding out of 13 commercial chiral columns. This means that the coated CSPs include more cellulose derivatives and are more frequently used for the resolution of chiral compounds than the bonded CSPs. Some of these chiral columns can be selectively used in normal-phase HPLC (NP-HPLC), like Chiralcel OD, Chiralcel OA, Chiralcel OB, Chiralcel OC, Chiralcel OF, Chiralcel OG and Chiralcel OJ etc.; some can be used in reversed-phase HPLC (RP-HPLC), like Chiralcel OD-R, Chiralcel OZ-R and Chiralcel OJ-R; and some can be used in both NP-HPLC and RP-HPLC, like Lux Cellulose-1, Lux Cellulose-2 , Lux Cellulose-3, Lux Cellulose-4, Chiralpak IB and Chiralpak IC [9,10]. Some studies have been done to evaluate comparatively the enantioselective and chromatographic properties of Chiralcel OD and Chiralpak IB using a set of 48 compounds that differ in their physical and chemical properties [11]. The uses of these CSPs in different mobile phases mainly depend on their different preparation methods.

216 Cellulose – Medical, Pharmaceutical and Electronic Applications

methods and evaluate their bioactivity and environmental fates.

**2. The development of cellulose-based CSPs** 

preparative large-scale separation [7].

phases or sample dissolving reagents [8].

and their applications in stereoselective separations of chiral pesticides.

Chiral compounds account for 25% of all agrochemical compounds used commercially and for 26% of the total value of the world agrochemical market [5]. The enantiomers of chiral pesticides possess similar physicochemical properties in a non-chiral environment while they show different activities in biological systems due to enantioselective interactions with enzymes, receptors, and other enantiomeric biological entities. For example, triadimenol is a systemic fungicide and has four stereisomers due to the presence of two chiral centers in its molecule. Of the four, the (1S, 2R)-isomer shows the highest fungicidal activity (up to 1000 fold more active than the other three) [6]. However, most chiral pesticides are produced and formulated as racemic mixture even though the desired biological activity may be derived from only one enantiomer. It is therefore very important to be able to separate enantiomers of chiral pesticides in order to prepare single enantiomers, develop enantiomeric analysis

This work focuses mainly on a review of the development of cellulose derivatives for CSPs which are prepared as cellulose-based chiral columns by coating and bonding on supports,

The cellulose-based CSPs generally are of two types: the coated and the bonded. The coated cellulose-based CSPs consisting of the low-molecular-weight cellulose benzoate or phenyl carbamate showed higher chiral recognition than the covalently bonded CSPs for most racemates. The major reason was considered to be an optimal secondary and supermolecular structure for the chiral recognition mechanism of polysaccharide derivatives under coated conditions [1,3]. However, the coated CSPs can only be used with a limited range of solvents as mobile phases such as alkanes, alcohols, acetonitrile, or aqueous solvents including alcohols or acetonitrile because CSPs may dissolve in 'strong' solvents such as tetrahydrofuran (THF) and chloroform (CHCl3). Such a dissolution would damage or destroy the CSPs. This limited the application range of the coated CSPs on separation and preparation of chiral compounds, because the solubility of the sample in the mobile phase is very important to increase the amount of racemates loaded on CSPs, especially on a

The bonded CSPs were prepared by covalently bonding cellulose derivates to silica gel. They can be applied to a wider range of resolving conditions than the coated type. The fixation can affect the conformation of cellulose derivates and make it difficult to obtain optimal supermolecular structure. This results in lower chiral recognition ability of the bonded-type CSPs. However, the fixation improves versatility in the solvent selection, and allows the use of some solvents that cannot usually be applied on the coated CSPs as mobile

The commercial cellulose-based CSPs including the coated and the bonded CSPs currently in use are summarized in Table 1. As can be seen, there are only two columns (Chiralpak IB and Chiralpak IC) prepared from cellulose derivatives by bonding out of 13 commercial chiral columns. This means that the coated CSPs include more cellulose derivatives and are


218 Cellulose – Medical, Pharmaceutical and Electronic Applications


The Development and Application of Cellulose-Based Stationary Phases

The investigations of four regioselectively substituted cellulose derivatives having two different substituents at 2-, 3-, and 6-positions showed better enantioseparations were sometimes obtained on these CSPs, compared to the corresponding homogeneously trissubstituted cellulose derivatives-based CSPs. Cellulose 2,3-(3-chloro-4 methylphenylcarbamate)-6-(3,5- dimethylphenylcarbamate), and 2,3- (3,5-dimethylphenylcarbamate)-6-(3-chloro-4-methylphenylcarbamate) exhibited the most efficient enantioseparations for tested racemates in four CSPs [14]. The cellulose derivative of benzoylcarbamate also showed a higher chiral discrimination ability compared to those of phenylcarbonate, *p*-toluenesulfonylcarbamate, and benzoylformate when they used as CSPs on HPLC. This discrimination could be achieved by hydrogen bonding of the racemates'

Chiral recognition abilities of cellulose-methoxyphenylcarbamates were significantly influenced by the position, bulkiness, and number of alkoxy groups introduced on the phenyl group. The 3-position was found to be the best for introducing an alkoxy group, and cellulosetris-(3-methoxyphenylcarbamates) exhibited much higher recognitions. Additionally, the recognition abilities also increased with the increases of the bulkiness of the 3-alkoxy group [16]. Cellulose-tris- (3-trifluoromethylphenylcarbamate) also exhibited characteristic enantioseparation and were better to resolve some chiral compounds than Chiralcel OD [17 ]. During the preparation of polymer cellulose-based CSPs by coating on silica gel, chiral additives such as (+)-L-Mandelic acid, (+)-1-phenyl-1,2-ethanediol and (-)-2-phenyl-1 propanol for CSPs of cellulose tribenzoate, and (-)-2-phenyl-1- propanol and (+) phenylsuccinic for CSPs of cellulose trisphenylcarbamate have a substantial effect on the resolution and efficiency of the CSPs, and can improve chiral recognition ability compared

Some new supports other than decorative silica gel were also used to prepare the coated CSPs. For example, a new CSP of CDMPC was prepared by coating CDMPC on TiO2/SiO2 particles. Its good chiral separation ability and a comparably low column pressure proved that TiO2/SiO2 could be used as an alternative to silica gel, and could enlarge the range of

CDMPC and CDCPC were covalently bonded to decorative silica gel to obtain the bonded chiral columns of Chiralpak IB and Chiralpak IC respectively [9]. CTPC regioselectively bonded at the 6-position to silica gel exhibited a higher chiral recognition than either CTPC regioselectively bonded at the 2- or 3-position or non-regioselectively bonded at the 2-, 3-, and 6-positions [20]. When cellulose derivatives bearing pyridyl and bipyridyl residues were compared in chiral recognition abilities, the results showed that the regioselectively substituted derivatives exhibited higher recognition compared with cellulose derivatives bearing these residues at the 2-, 3- and 6-positions of a glucose ring. This ability was significantly influenced by the coordination of Cu(II) ion to the bipyridyl groups that

resulted in the difference of the higher-order structures of cellulose derivatives [21].

hydrogen atoms with the carbonyl group of the benzoylcarbamates [15].

to the original CSPs [18].

base materials when preparing CSP [19].

**2.2. The development of bonded cellulose-based CSPs** 

in Stereoselective Separation of Chiral Pesticides 219

**Table 1.** The list of commercial cellulose-based CSPs in the present.

## **2.1. The development of coated cellulose-based CSPs**

Various cellulose derivatives were reported as CSPs in recent years, especially on cellulose benzoates and phenylcarbamates because of their higher enantiomeric discrimination ability and wide applications. Okamoto et al, synthesized some cellulose triphenylcarbamate derivatives and absorbed them on silica gel as CSPs, and then compared optical resolution abilities with the characteristics of the substituents on the phenyl rings. The results showed that dimethylphenyl- and dichlorophenylcarbamates substituted at 3,4- or 3,5-positions exhibited better chiral recognition for most reacemates than monosubstituted derivaties. Of the these, cellulose tris-(3,5-dimethylpheyl-carbamate) (CDMPC) offered the highest enantiomeric separability [12]. In another investigation on chiral recognition ability of cellulose phenylcarbamate derivatives, cellulose-tris-(3-fluoro-5-methylphenylcarbamate) was reported to be better than 3,5-difluoro- and 3,5-dimethylphenylcarbamates of cellulose for enantioseparation of ten racemates [13].

The investigations of four regioselectively substituted cellulose derivatives having two different substituents at 2-, 3-, and 6-positions showed better enantioseparations were sometimes obtained on these CSPs, compared to the corresponding homogeneously trissubstituted cellulose derivatives-based CSPs. Cellulose 2,3-(3-chloro-4 methylphenylcarbamate)-6-(3,5- dimethylphenylcarbamate), and 2,3- (3,5-dimethylphenylcarbamate)-6-(3-chloro-4-methylphenylcarbamate) exhibited the most efficient enantioseparations for tested racemates in four CSPs [14]. The cellulose derivative of benzoylcarbamate also showed a higher chiral discrimination ability compared to those of phenylcarbonate, *p*-toluenesulfonylcarbamate, and benzoylformate when they used as CSPs on HPLC. This discrimination could be achieved by hydrogen bonding of the racemates' hydrogen atoms with the carbonyl group of the benzoylcarbamates [15].

218 Cellulose – Medical, Pharmaceutical and Electronic Applications

ed name

9 cellulose-*tris*-acetate CTA Chiralcel OA Coating

Bonded CDMPC

Bonded CDCPC

**Table 1.** The list of commercial cellulose-based CSPs in the present.

**2.1. The development of coated cellulose-based CSPs** 

for enantioseparation of ten racemates [13].

Commercial product

MCTA Chiralcel CA-1 Coating

CTC ChiralcelOK Coating

Chiralpak IB Bonding

Chiralpak IC Bonding

Various cellulose derivatives were reported as CSPs in recent years, especially on cellulose benzoates and phenylcarbamates because of their higher enantiomeric discrimination ability and wide applications. Okamoto et al, synthesized some cellulose triphenylcarbamate derivatives and absorbed them on silica gel as CSPs, and then compared optical resolution abilities with the characteristics of the substituents on the phenyl rings. The results showed that dimethylphenyl- and dichlorophenylcarbamates substituted at 3,4- or 3,5-positions exhibited better chiral recognition for most reacemates than monosubstituted derivaties. Of the these, cellulose tris-(3,5-dimethylpheyl-carbamate) (CDMPC) offered the highest enantiomeric separability [12]. In another investigation on chiral recognition ability of cellulose phenylcarbamate derivatives, cellulose-tris-(3-fluoro-5-methylphenylcarbamate) was reported to be better than 3,5-difluoro- and 3,5-dimethylphenylcarbamates of cellulose

Coating

Type Chemical structure of cellulose derivative

O

O

O

O

O OCOCHCH

CH3

CH3 CH3

CH3

HCHCOCO OCOCHCH

O

OCONH

O

Cl

Cl Cl

Cl

HNOCO OCONH

O

O OCONH

HNOCO OCONH

OOC

O

COO OOC

O

O

OOCCH3

OOCCH3

H3CCOO OOCCH3

H3CCOO OOCCH3

H3C

H3C

Cl

Cl

[9,10]

CTB Chiralcel OB-H ChiralcelOB

No. Chemical name Shorten-

8 cellulose-*tris*benzoate

10 Mricocrystalline

11 cellulose-*tris*cinnamate

12 cellulose-*tris*-(3,5 dimethylphenylcarba

13 cellulose-*tris*-(3,5 dichloro-

phenylcarbamate)

mate)

cellulose-*tris*-acetate

Chiral recognition abilities of cellulose-methoxyphenylcarbamates were significantly influenced by the position, bulkiness, and number of alkoxy groups introduced on the phenyl group. The 3-position was found to be the best for introducing an alkoxy group, and cellulosetris-(3-methoxyphenylcarbamates) exhibited much higher recognitions. Additionally, the recognition abilities also increased with the increases of the bulkiness of the 3-alkoxy group [16]. Cellulose-tris- (3-trifluoromethylphenylcarbamate) also exhibited characteristic enantioseparation and were better to resolve some chiral compounds than Chiralcel OD [17 ].

During the preparation of polymer cellulose-based CSPs by coating on silica gel, chiral additives such as (+)-L-Mandelic acid, (+)-1-phenyl-1,2-ethanediol and (-)-2-phenyl-1 propanol for CSPs of cellulose tribenzoate, and (-)-2-phenyl-1- propanol and (+) phenylsuccinic for CSPs of cellulose trisphenylcarbamate have a substantial effect on the resolution and efficiency of the CSPs, and can improve chiral recognition ability compared to the original CSPs [18].

Some new supports other than decorative silica gel were also used to prepare the coated CSPs. For example, a new CSP of CDMPC was prepared by coating CDMPC on TiO2/SiO2 particles. Its good chiral separation ability and a comparably low column pressure proved that TiO2/SiO2 could be used as an alternative to silica gel, and could enlarge the range of base materials when preparing CSP [19].

## **2.2. The development of bonded cellulose-based CSPs**

CDMPC and CDCPC were covalently bonded to decorative silica gel to obtain the bonded chiral columns of Chiralpak IB and Chiralpak IC respectively [9]. CTPC regioselectively bonded at the 6-position to silica gel exhibited a higher chiral recognition than either CTPC regioselectively bonded at the 2- or 3-position or non-regioselectively bonded at the 2-, 3-, and 6-positions [20]. When cellulose derivatives bearing pyridyl and bipyridyl residues were compared in chiral recognition abilities, the results showed that the regioselectively substituted derivatives exhibited higher recognition compared with cellulose derivatives bearing these residues at the 2-, 3- and 6-positions of a glucose ring. This ability was significantly influenced by the coordination of Cu(II) ion to the bipyridyl groups that resulted in the difference of the higher-order structures of cellulose derivatives [21].

CSP with poly[styrene-*b*-cellulose 2,3-bis-(3,5-diphenylcarbamate)] was prepared by the surface-initiated atom transfer radical polymerization (SI-ATRP) of cellulose 2,3-bis-(3,5 dimethylphenylcarbamate)-6-acrylate after the SI-ATRP of styrene on the surface of silicon dioxide supports in pyridine. This CSP showed considerably high column efficiency for the resolution of tested racemates [22].

The Development and Application of Cellulose-Based Stationary Phases

O

HNOCO OCONH

<sup>R</sup> <sup>R</sup>

OCONH

OOC

O

O

R

R

COO OOC

<sup>C</sup> <sup>O</sup> <sup>O</sup>

Investigations on the influence of the pore size of silica gel, the coating amount , the coating solvent, and the column temperature on chiral discrimination of CDMPC showed that CSPs prepared with a large-pore silica gel having a small surface area exhibited higher recognition abilities. An increase in the amount of coating of CDMPC on the silica gel can improve the loading capacity of racemates, and a CSP coated with 45% CDMPC by weight can be used for both analytical scale and semi-preparative scale separations. CSPs coated with acetone showed higher enantioselectivity than those coated with THF or a mixture of

Generally, cellulose-derived CSPs covalently bonded on silica gel are prepared by using a benzoyl chloride or a phenyl isocyanate to react with cellulose in homogeneous conditions, to obtain the corresponding benzoates or carbamates. However, other methods to prepare this type of CSP have been reported. Ikai et al. summarized various immobilization methods of the polysaccharide derivatives mainly onto silica gel: immobilization using diisocyanate, vinyl groups by polymerization and copolymerization with a vinyl monomer etc. [28,29].

CDMPC can be efficiently immobilized on silica gel as CSPs by copolymerizing with vinyl monomers. The introduction of vinyl groups or the employment of vinyl monomers can readily tune the immobilization efficiency and the chiral recognition of cellulose derivatives [30]. The new method was applied to immobilize CDMPC onto bare silica gel via the intermolecular polycondensation of triethoxysilyl groups, which were introduced onto the

<sup>R</sup> <sup>R</sup>

CSPs. The APS was prepared beforehand by silanizing silica gel with a solution of 3 aminopropyltriethoxysilane. Finally, the CSPs were packed into HPLC columns by the slurry method, to obtain coated chiral columns [18, 26]. For example, CDMPC was synthesized by reaction of microcrystalline cellulose with 3,5-dimethylphenylcarbimide in pyridine; the product was filtered off, washed with methanol and dried at 60° C for 24h. CDMPC was then dissolved in THF and coated on the APS under vacuum to dryness. Finally, the coated CDMPC were packed into a stainless-steel column at 3.7×107Pa by the

high-pressure slurry method to obtain the corresponding CSP [26].

R

C

pyridine

N

pyridine

**Figure 1.** The synthesized routes of cellulose benzoates or phenylcarbamates.

**3.2. The preparation method of covalently bonded CSPs** 

Several methods of synthesis are shown in Figures 2 to 4.

Cl O

O

OH

O

R

HO HO

CH2Cl2 and phenol [27].

in Stereoselective Separation of Chiral Pesticides 221

Laureano Oliveros et al, prepared five mixed 10-undecenoate/benzoates of cellulose and linked them to allyl silica gel by means of a radical reaction. The investigation of chiral recognition ability showed that CSP5 (10-undecenoate/3,5-dichlorobenzoate) has the highest enantioselectivity for most of tested racemates, followed by CSP3 (10-undecenoate/4 methylbenzoate) and CSP4 (10-undecenoate/benzoate). These CSPs showed lower resolution than the coated CSPs although they have higher column efficiency. The reason may be the lack of polar amino groups on the surface of the CSPs. However, when being compared with the coated CSPs, these CSPs can tolerate the use of more polar solvents such as chloroform in the mobile phase [23].

Three cellulose-based CSPs were prepared by reticulation of the same cellulose derivative on three end-capped silica gels with different pore sizes (50Å, 100Å and 4000Å). The comparison of chiral recognition ability among them showed that CSPs with higher pore size exhibited higher selectivity factors, because it can accommodate a larger amount of accessible cellulose derivative on its surface [7].

Four mixed 10-undecenoyl-3,5-dimethylphenylaminocarbonyl derivatives of cellulose with increased proportion of alkenoyl groups were bonded on allylsilica gel. Their comparison showed that CSPB presents the best chiral recognition and can separate the widest range of the tested racemates. The reason may be the higher number of substitution of glucose units. The important decrease in the recognition ability of these CSPs could be attributed to their higher degree of reticulation. More heterogeneous reaction sites of allysilica gel with cellulose derivatives can result in lower degree of reticulation in CSPs and therefore improve their recognition ability [24].

Azido cellulose phenylcarbamate (AzCPC) was synthesized regioselectively and chemically immobilized onto amino-functionalized silica gel to obtain urea-bonded CSPs. Enantioseparation using CHCl3 on these CSPs showed better separation than traditional hexane/2-propanol in mobile phases for some tested racemates. The pre-coating of AzCPC onto silica gel prior to chemical immobilization could significantly improve immobilization efficiency, and obtained better enantioselectivity [25].
