Separation of structural isomers by "in-situ" modified monolithic columns with b-cyclodextrin

Abstract  Reactive continuous rods of macroporous poly (glycidyl methacrylate-co-ethylene dimethacrylate) have been prepared by "in-situ" copolymerization of the monomers within the confines of a 150×4.6mm I.D. chromatography column in the presence of porogenic diluent, and b-cyclodextrin was immobilized on the continuous rods. The effect of the flow rate on the back pressure drop of the continuous rods was also investigated, which showed that the back pressure was greatly reduced when comparing to the classical column. By using a pair of nitrophenol isomeric compounds as the targets, the modified monolithic column was investigated and good resolution was obtained.
Keywords continuous rods;b-cyclodextrin;separation isomers

1. INTRODUCTION
Chiral stationary phases based on proteins, cyclodextrins, cellulose derivatives and macrocyclic antibiotics are widely used in the separation of chiral compounds by liquid chromatography. These chiral ligands are usually immobilized on silica material. However, conventional commercial columns packed with beads in HPLC possess some inherent limitations such as the slow diffusional mass transfer and the large void volume between the packed particles. Although some new stationary phases such as the non-porous beads ^[1,2] and perfusion chromatography packings ^[3,4] are designed to resolve these problems, these limitations cannot be overcome in essence. In recent years a novel type, the monolithic stationary phase, has attracted increasing attention in liquid chromatography because of its easy preparation, excellent properties and high performance compared to conventional columns packed with particles for the separation of biopolymers.The monolithic columns have been prepared for the reversed-phase chromatography ^[5,6], ion-exchange chromatography ^[7,8], hydrophobic interaction chromatography ^[9,10], affinity chromatography ^[11,12], high throughput bioreactors chromatography^[13,14]. However, similar chiral stationary phases based on monolithic rods have not been reported so far. The only example of the separation of chiral compounds is in monolithic molecularly imprinted polymers ^[15].b-cyclodextrin is a chiral ligand, it has been used extensively for enantiomeric and diastereomeric separations by HPLC and nitrophenol isomers were often used to investigate the characteristic of the chemically bonded b -cyclodextrin stationary phase for HPLC^[16,17]. The article (Reference 17) described the synthesis of b-cyclodextrin -copolymers coated on silica beads and used for the separation of nitrophenol isomers for HPLC. The isomers could be partly separated, but the resolution was not satisfactory. This report dealt with how b -cyclodextrin was immobilized on the monolithic rods and its performance for the separation of a pair of nitrophenol isomeric compounds.

2. EXPERIMENTAL
2.1 Chemicals
Glycidyl methacrylate(Suzhou Anli Chemicals Co. Ltd.), ethylene dimethacrylate(New Jersey, USA ), azobisisobutyronitrile( The Fourth Factory of Shanghai Reagent), dodecanol, cyclohexanol，b-cyclodextrin, (Shanghai Chemical Reagent Company of China Medicine), H₂SO₄, ammonium dihydrogen phosphate (Factory of Baoding Chemical Reagent).
2.2 Preparation of the monolithic column
The continuous column was prepared by in situ polymerization of monomers within the confines of the stainless-steel tube of a 150×4.6 mm I.D. chromatographic column. First, a mixture consisting of 2.2 mL glycidyl methacrylate, 1.5mL ethylene dimethacrylate(monomers), 0.8 mL dodecanol, 7.0mL cyclohexanol(porogenic diluents), 0.09g azobisisobutyronitrile(initiator) was purged with nitrogen for 10min. Then the stainless steel tube sealed at the bottom was filled with the polymerization mixture and then sealed at the top. After the polymerization was allowed to proceed at 55^oC for 24h, the seals were removed from the tube and the column was provided with fittings, attached to the HPLC system and washed with tetrahydrofuran(THF) at a flow-rate of 1mL/min for 60min to remove the alcohols and other soluble compounds present in the polymer rod after the polymerization was completed.
2.3 Preparation of b -cyclodextrin-functionalized porous monolith
The epoxide groups of the poly (glycidyl methacrylate-co-ethylene dimethacrylate) were owned to react with b-cyclodextrin. The amount of the immobilized b-cyclodextrin was determined as described in the literature^[18]. Using the optimum conditions, the solution that b-cyclodextrin resolved in the solution of sodium hydroxide was pumped through the column at a flow-rate of 0.1mL/min. The modified columns were then washed with water at a flow-rate of 1mL/min for 2h.
2.4 Chromatography and Instrument
The chromatography system consisted of JASCO (JASCO, Japan) PU-1580 and PU-1586 pumps, a variable wavelength UV-1570 detector. Data processing was carried out with a JASCO LC-1500 workstation. Mobile phases were prepared by mixing methanol and acetonitrile with solution of 50mmol/L ammonium dihydrogen phosphate (7:1:2, v/v/v, pH was adjusted to 4.0 with phosphoric acid). UV3000 spectro-meter (Shimadzu).

3. RESULTS AND DISCUSSION
3.1 Functionalization of the monolithic columns
The amount of b -cyclodextrin immobilized on the monolithic column was affected by several factors, such as temperature, time of reaction, the concentration of sodium hydroxide. In order to investigate the effect of these factors on the immobilized amount of b -cyclodextrin, immobilization process were investigated outside of the column. When b -cyclodextrin immobilized on the polymer was hydrolyzed by H₂SO₄ solution, glucose would produce. By detecting glucose at 490 nm using spectro-meter,   the optimum condition could be obtained. This was because that the larger amount of b -cyclodextrin was immobilized, the higher absorbance was obtained. First, we have investigated the effect of the concentration of sodium hydroxide on the immobilized amount of b -cyclodextrin. At the beginning, the absorbance increased when the concentration of sodium hydroxide increased, when the concentration of sodium hydroxide exceeded 0.1 mol/L, the absorbance decreased. So 0.1 mol/L sodium hydroxide was chosen. Through the same way, optimum temperature and reaction time were also obtained. So the optimum conditions immobilizing b -cyclodextrin on monolithic column were as follows: temperature was 55^oC, time was 5h and the concentration of sodium hydroxide was 0.1mol/L. Under this condition, the monolithic column was modified by b -cyclodextrin.
3.2 Separation of aromatic compounds
In order to achieve optimum resolution for m- and o-nitrophenol, the effects of the composition of the mobile phase and pH were investigated. First, we have investigated a series of mixture of methanol and ammonium dihydrogen phosphate solution. When decreased the ratio of methanol, both t_R of m- and o-nitrophenol increased, but the maximum resolution was below 1. At the optimum ratio, the effect of pH on the resolution was studied, the results showed that pH 4.0 is better than others, but the resolution still could not exceed 1. So we added acetonitrile into the mixture mobile phase when the ratio of water kept unchanged. The experiments showed that the nitrophenol isomers could obtain better separation on the b -cyclodextrin modified monolithic column when the mobile phase composition was a ternary solvent mixture composed by acetonitrile-methanol-50mmol/L ammonium dihydrogen phosphate solution, pH 4.0(7:1:2,v/v/v). The chromatogram was shown in Figure 1.

Fig. 1 Separation of nitrophenol isomers on b -cyclodextrin modified monolithic column
1：m-nitrophenol；2：o-nitrophenol

4. CONCLUSIONS
The results proved that b -cyclodextrin can be successfully immobilized on the monolithic column and the modified column can be used for the separation of isomers. It means that other chiral ligands are also possible to be immobilized on the monolithic column for separation purpose.

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