Preparation Of β-cyclodextrin Bonded Monolithic Column For Rapid Separation And Determination Of Puerarin From Kudzu Flavonoids

Monolithic column is a new technology, which can be prepared by in-situ polymerizaton using functional monomers, cross-linkers, pore-forming agents and initiators. It has already exhibited some advantages, such as easy preparation and modification, low column pressure, rapid mass transfer and high separation efficiency.β-cyclodextrin (β-CD) has a special cavity structure, so it can choose and identify various guest molecules to form supramolecular inclusion compound. This thesis synthesized acryloyl-monolith and poly(glycidyl methacrylate-ethylene glycol dimethacrylate) (poly(GMA-EGDMA)) monolithic skeleton, choosingβ-CD and its derivatives as modifiers, then the reaction and modify conditions were optimized and the monoliths were used to separate and purify natural extracts of kudzu flavonoids. Molecular dynamics modeling has been used to study the separation and recognition mechanisms of pueraria flavonoids onβ-CD functionalized monolith. The main research work were as follows: 1. Acryloyl-monolith was prepared to separate and purify natural extracts of kudzu flavonoids. This monolith was prepared in a stainless steel chromatographic column tube (100 mm×4.6 mm i.d.) by co-polymerisation of acryloyl-β-CD and ethylene glycol dimethacrylate (EGDMA), with 2,2'-azobisisobutyronitrile (AIBN) as initiator, and dimethyl sulfoxide (DMSO) as pore-forming agent. The optimal synthetic conditions were:acryloyl chloride:β-CD=1:1.5 (molar ratio), acryloyl-β-CD:EGDMA=1:2 (molar ratio), synthetic temperature is 60℃.The average pore size of the resulting monolith was 1.6μm,specific surface area was 11.5 m/g, and its total porosity was 57.6%. With 12% acetic acid solution as the mobile phase, acryloyl-monolith could well separate and purify puerarin from natural extracts of kudzu flavonoids. The purity of the product puerarin was 93%, and the recovery was 76%.2. The poly(GMA-EGDMA) monolithic skeleton was prepared on 65℃. The functional monomer and cross-linker were GMA and EGDMA, initiator was AIBN, and porogenic agent was toluene/heptane (4/1 v/v). The monolithic skeleton was modified by EDA-β-CD on 60℃for 12h. The average pore size of the functionalized monolith was 0.48μm, specific surface area was 9.85 m2/g, and its total porosity was 55.4%. It has done a good job in separating and purifing puerarin from natural extracts of kudzu flavonoids, with 40% acetic acid solution as the mobile phase. The purity of the product puerarin was 95%, and the recovery was 83%.3. Theβ-CD and oligo-β-CD instead of CD derivatives were used as modified compounds.The best conditions for modification were:the monolith was grafted by oligo-β-CD (0.7 mol/L) for 5 times in 0.2M Na2CO3/NaHCO3(PH=10) aqueous solution, and each time interval was 3-4h. The average pore size of the resultant monolith was 0.92μm, specific surface area was 5.25 m2/g, and its total porosity was 59%. The oligo-β-CD modified monolith could efficiently separate the mixture of puerarin, daidzin and daidzein, the separation factors were 0.903 and 1.955. Meanwhile, the purity of the product puerarin separated from natural extracts of kudzu flavonoids was 95%, and the recovery was 83%.