**3. The preparation method of cellulose-based CSPs**

## **3.1. The preparation method of coated CSPs**

Generally, benzoate and phenylcarbamate derivatives of cellulose were prepared by reaction between cellulose and excess benzoyl chloride or phenyl isocyanate derivatives in dry pyridine (Figure 1). These derivatives are then coated onto macro-porous 3 aminopropylsilica (APS) from a solution by evaporation of the solvent to obtain coated 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].

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

220 Cellulose – Medical, Pharmaceutical and Electronic Applications

resolution of tested racemates [22].

as chloroform in the mobile phase [23].

improve their recognition ability [24].

efficiency, and obtained better enantioselectivity [25].

**3.1. The preparation method of coated CSPs** 

**3. The preparation method of cellulose-based CSPs** 

accessible cellulose derivative on its surface [7].

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

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

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

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

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

Generally, benzoate and phenylcarbamate derivatives of cellulose were prepared by reaction between cellulose and excess benzoyl chloride or phenyl isocyanate derivatives in dry pyridine (Figure 1). These derivatives are then coated onto macro-porous 3 aminopropylsilica (APS) from a solution by evaporation of the solvent to obtain coated 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 CH2Cl2 and phenol [27].

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

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

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

glucose unit by the epoxide ring-opening reaction under acidic conditions. The CSPs thus obtained also exhibited high chiral recognition ability for 10 tested racemates and could be used with various eluents that are not compatible with the conventionally coated CSPs [31]. One-pot method was applied to synthesize CDCPC bearing a small amount of 3- (triethoxysilyl) propyl residues, and then immobilized onto silica gel through intermolecular polycondensation. The immobilized CSPs exhibited chiral recognition abilities similar to the corresponding coated CSP and slightly different from the commercial Chiralpak IC [32].

The Development and Application of Cellulose-Based Stationary Phases

O

OH

coating

O

functionalities are not observed. [37].

**pesticides** 

n

1)

C Cl

HO HO

C O toluene/pyridine (5:1)

CSPs, and a lower amount can produce higher resolution ability [36].

resolution results of 79 chiral pesticides in current references.

O C N N C O

CH3

2) H3C <sup>N</sup>

O

OTr

not isolated

**Figure 4.** Regioselective covalent bonding of CDMPC to position 6 of the glucosidic rings [34].

O

n

O Si <sup>O</sup> <sup>N</sup> H

O

Cellulose-(diphenymethyldicarbamate/phenylcarbamate) covalently bonded to APS showed some chiral recognition ability [35]. Cellulose-tris-phenylcarbamate was covalently bonded to silica gel with different spacers. The results showed CSPs prepared with spacer 1(4-(1-(3- (triethoxysilyl)-propyl)urea)-benzyl-4-isocyanatobenzene) exhibited higher resolution ability than spacer TEPI (3-(triethoxysilyl) propyl isocyanate) with the same preparation procedure. The amount of spacer in the synthesis influences the optical resolution ability of

Polar monodisperse amine terminated polymer (2-aminoethyl methacrylate-coethylenedimethacrylate) beads can be used as the replacement of silica gel, and are suitable as supports for the preparation of cellulose-based CSPs coated by simple adsorption and immobilized with a diisocyanate linker. However, the chiral recognition abilities of these CSPs shows no enhancement because the uses of cellulose-based selectors and preparation methods may completely cover the surface of polymer supports. Thus, the analytes have no access to the native surface of the support and non-specific interactions with the surface

**4. The application of cellulose-based CSPs in enantioseparation of chiral** 

Chiral HPLC is a good method to separate enantiomers/stereoisomers of chiral pesticides because it facilitates the preparation of single enantiomers for study of enantiomeric bioactivity, toxicology and environmental fate. In recent years, cellulose-based CSPs prepared with different cellulose derivatives and methods resulted in their very broad application for chiral separation of pesticides such as organophosphates [38], organochlorine, triazole, synthetic pyrethroids, acylanilides, imidazolinones, phenoxypropanoic-acid herbicides and related compounds [39].Table 2 summarizes the

2) HCl/CH3OH

H3C N

O N

1) C O

CH3

HO HO

O Si <sup>O</sup> NH2

O

in Stereoselective Separation of Chiral Pesticides 223

O

OH

O

n

RO RO

> H O

RO RO O

O

O

n

<sup>H</sup> <sup>N</sup>

**Figure 2.** The covalent bonding of 3,5-dichloro- and 3,5-dimethylphenylcarbamate of cellulose onto APS [33].

**Figure 3.** Regioselective covalent bonding of CDMPC to positions 2 and 3 of the glucosidic rings.

O Si O

2) X N

X

O

1)

APS [33].

NH2

O C N N C O

C O

O

O C N N C O

CH3

CH3

OH

O

n

C O toluene/pyridine (5:1)

2) <sup>C</sup> <sup>O</sup> <sup>O</sup> Si <sup>O</sup> <sup>N</sup>

HO HO

2) H3C <sup>N</sup>

1) HCl/CH3OH H3C N

1)

X: Cl, CH3

toluene/pyridine (5:1) <sup>O</sup> Si

glucose unit by the epoxide ring-opening reaction under acidic conditions. The CSPs thus obtained also exhibited high chiral recognition ability for 10 tested racemates and could be used with various eluents that are not compatible with the conventionally coated CSPs [31]. One-pot method was applied to synthesize CDCPC bearing a small amount of 3- (triethoxysilyl) propyl residues, and then immobilized onto silica gel through intermolecular polycondensation. The immobilized CSPs exhibited chiral recognition abilities similar to the corresponding coated CSP and slightly different from the commercial Chiralpak IC [32].

O

O

C Cl

O

OTr

O

N H

**Figure 2.** The covalent bonding of 3,5-dichloro- and 3,5-dimethylphenylcarbamate of cellulose onto

HO HO

<sup>O</sup> Si <sup>O</sup> <sup>N</sup> H

O

O

**Figure 3.** Regioselective covalent bonding of CDMPC to positions 2 and 3 of the glucosidic rings.

O N H

O

O N

H

O N

OTr

O

n

O

<sup>H</sup> <sup>N</sup>

<sup>H</sup> <sup>N</sup>

<sup>n</sup> 3-aminopropylsilica gel coated with cellulose

> N H

coating

<sup>O</sup> Si <sup>O</sup> NH2

H O

RO RO

H O O

O

RO RO

O

O

OR

OTr

O

O

n

n

O

RO RO O

OR

O

n

HO HO

2) HCl/CH3OH

1) coating with

**Figure 4.** Regioselective covalent bonding of CDMPC to position 6 of the glucosidic rings [34].

Cellulose-(diphenymethyldicarbamate/phenylcarbamate) covalently bonded to APS showed some chiral recognition ability [35]. Cellulose-tris-phenylcarbamate was covalently bonded to silica gel with different spacers. The results showed CSPs prepared with spacer 1(4-(1-(3- (triethoxysilyl)-propyl)urea)-benzyl-4-isocyanatobenzene) exhibited higher resolution ability than spacer TEPI (3-(triethoxysilyl) propyl isocyanate) with the same preparation procedure. The amount of spacer in the synthesis influences the optical resolution ability of CSPs, and a lower amount can produce higher resolution ability [36].

Polar monodisperse amine terminated polymer (2-aminoethyl methacrylate-coethylenedimethacrylate) beads can be used as the replacement of silica gel, and are suitable as supports for the preparation of cellulose-based CSPs coated by simple adsorption and immobilized with a diisocyanate linker. However, the chiral recognition abilities of these CSPs shows no enhancement because the uses of cellulose-based selectors and preparation methods may completely cover the surface of polymer supports. Thus, the analytes have no access to the native surface of the support and non-specific interactions with the surface functionalities are not observed. [37].
