Preparation and Chiral Separation of a Novel Immobilized Cellulose-Based Chiral Stationary Phase in High-Performance Liquid Chromatography

Journal of Chromatographic Science 2012;50:516– 522 doi:10.1093/chromsci/bms047 Advance Access publication April 20, 2012 Article Preparation and Chiral Separation of a Novel Immobilized Cellulose-Based Chiral Stationary Phase in High-Performance Liquid Chromatography Gui Ming Peng1, Su Qin Wu1, Zhi Li Fang1, Wei Guang Zhang1*, Zhen Bin Zhang1, Jun Fan1, Sheng Run Zheng1, Shang Sen Wu1 and Siu Choon Ng1,2 1 School of Chemistry and Environment, South China Normal University, Guangzhou, 510006, China, and 2Division of Chemical and Biomolecular Engineering, Nanyang Technological University, 637459, Singapore *Author to whom correspondence should be addressed. Email: Received 15 July 2011; revised 13 September 2011 The chiral selector 6-azido-2, 3-di( p-chlorophenylcarbamoylated) cellulose was synthesized and further chemically immobilized onto 5-mm amino functionalized spherical porous silica gel. It was used as chiral stationary phase in high-performance liquid chromatography. Thirty racemates were successfully separated into enantiomers in either normal phase mode or reversed-phase mode. Good reproducibility and stability of the chiral stationary phase have been demonstrated. immobilized onto silica gel used as CSPs; for example, cellulose 3,5-dimethylphenylcarbamate (Chiralpak IB) (14), cellulose tris(3,5-dichlorophenylcarbamate) (Chiralpak IC) (19) and azido cellulose phenylcarbamate (20). Cellulose tris(p-chlorophenylcarbamate) used as chiral selector that was coated onto silica gel was reported by Okamoto et al. (5). In this paper, cellulose p-chlorophenylcarbamate was first chemically immobilized onto silica gel by Staudinger reaction. The enantioseparation results showed that the CSP afforded high enantioseparation ability towards structurally diverse chiral compounds in either normal or reversed-phase mode. Introduction Many drugs, natural products and food compounds are chiral, with their enantiomers often showing different or even opposing pharmacology, toxicity and metabolic activities (1). Currently, many top-selling drugs around the world are single enantiomers with the desired biological activity (2). Moreover, the obtainment of pure enantiomers from racemates is an important concern in the stereochemical area. Among many other methodologies, high-performance liquid chromatography (HPLC) using chiral stationary phase (CSP) affords one of the most direct and effective approaches for enantioseparation. During the past two decades, CSPs have advanced rapidly. The majority of polysaccharide-based CSPs employed have been cellulose-based columns (3). Polysaccharide derivatives that were coated on silica gel as CSPs appeared in the 1980s (4, 5). However, solvents that can swell or dissolve the derivatives could not be used as mobile phases, because the chiral selectors were physically coated onto the surface of the silica gel. Accordingly, coated CSPs were only amenable for use in a limited range of eluents, which were usually mixtures of nonpolar solvents and alcohols used in normal phase mode (6, 7). To overcome the solubility of the coated selectors, Okamoto et al. first bonded the polysaccharide to a g-aminopropyl silica gel matrix using a diisocyanate as a spacer that was expected to react with the free amino groups on the matrix surface and the hydroxyl groups of the polysaccharide (8). Because polysaccharide derivatives were covalently bonded onto silica gel, the CSP could be applied with a much broader range of solvents as mobile phases (9 –17), which would enhance the success rate in enantioseparation. As for amylose-based CSPs, Chiralpak IA is a most successful immobilized CSP using amylose 3,5-dimethylphenylcarbamate as its chiral selector (18). Thus far, many functional groups have been used to modify cellulose and have been further Materials and Methods Equipments Nuclear magnetic resonance (NMR) was carried out on a Bruker ACF300FT-NMR spectrometer with tetramethylsilane as internal standard. Fourier transform infrared (FTIR) was performed on a Bio-Rad TFS156 instrument. Elemental analysis was determined on a PerkinElmer 2400CHN analyzer. The columns were packed using an Alltech pneumatic HPLC pump. Evaluation of the column was performed on an HPLC system, which comprised a Lib Alliance HPLC Series iii system, a Lib Alliance Model 201 ultraviolet-visible (UV-vis) detector and a 7725i injector equipped with a 20-mL sample loop. Chemicals and reagents Microcrystalline cellulose [degree of polymerization (DP)  200] and p-chlorophenyl isocyanate were purchased from Shanghai Hengxin Chemical Reagent Co.3-Aminopropyltriethoxysilane and Compounds 9, 12-15, 20-22, 24, 29 and 30 were obtained from Alfa Aesar (Tianjin, China); Compounds 1–8 were provided by Professor Ding-Qiao Yang’s lab, and the other compounds were obtained from Professor Zhao-Yang Wang’s lab. HPLC-grade hexane, acetonitrile, isopropyl alcohol (IPA), methanol and ethanol were purchased from Tianjin Damao Chemical Reagent Co. Deionized and distilled water was used throughout the study. Silica gel (5 mm, 500 Å, 300 m2/g) was purchased from Fuji Silysia Chemical Ltd. (Aichi, Japan). Preparation of mobile phases and samples Triethylammonium acetate buffer (TEAA) was prepared by adding acetic acid to a solution containing 0.1% (v %) of # The Author [2012]. Published by Oxford University Press. All rights reserved. For Permissions, please email: Figure 1. Synthetic procedures of the CSP. NaN3/dry DMSO/1008C (A); p-chlorophenyl isocyanate/dry pyridine/triethylamine/1008C (B); amino functionalized silica gel/PPh3/ THF/carbon dioxide (C). triethylamine to adjust to the desired pH 4.0. NaClO4 aqueous solution was controlled at the concentration 0.30 mol /L. All buffers were filtrated through a 0.45-mm membrane and degassed before use. The samples were prepared at a concentration of approximately 1 mg/mL. Ten microliters of the sample was injected. All chromatographic experiments were carried out at room temperature. Preparation of CSP Figure 1 shows the synthetic route to the CSP. Preparation of 6-azido-deoxycellulose (Compound B) Microcrystalline cellulose was functionalized with a p-toluene sulfonylchloride in N, N-dimethylacetamide/LiCl system to afford 6-tosyl-cellulose (Compound A) according to the reported method (21). FTIR (cm21, KBr): 3524 (OH), 3066 (C-Harom), 1598, 1496, 1455 (C ¼ Carom), 1367, 1176 (-SO2-), 1060 (C-O-C). Elemental analysis, found: C 49.85%, H 4.97%, S 10.39%. Degree of substitution was 1.06, which was calculated by using sulphur content. This indicated that almost all 6-OH and partial 2-OH or 3-OH of cellulose were converted to tosyl. Pre-dried Compound 1 (3.16 g) was dissolved in dry DMSO (100 mL). After addition of NaN3 (3.25 g), the mixture was stirred at 1008C for 24 h in a nitrogen atmosphere. Then the product was cooled to room temperature and separated by precipitation in ice-cold water (1,500 mL), filtered off and washed with distilled water and eth (...truncated)