Preparation And Application Of Kaempferol Molecularly Imprinted Monolithic Column

The molecularly imprinted monolithic column combines the advantages of high selectivity of molecularly imprinted polymer（MIPs）and high permeability of monolithic column and has been widely used in the separation and analysis of natural products.The preparation of molecularly imprinted monolithic columns by surface imprinting method using inorganic monolithic columns with optimized pore structure as the carrier is expected to overcome some shortcomings that currently exist in molecularly imprinted monolithic columns,such as simplifying the optimization process of pore structure,improving the imprinting efficiency,increasing the surface uniformity,improving the anti-swelling and shrinkage ability and increasing the stability of chromatographic columns.The purpose of this study is to prepare molecularly imprinted monolithic columns with high selectivity,high permeability,high efficiency and high stability,using kaempferol as template molecules and silica monolithic columns with uniform pore structure as support.This study provides new materials,new methods and basic experimental data for rapid and efficient separation of target products from complex natural product systems.The main research contents of this paper are as follows:（1）Kaempferol-MIPs were screened in detail and applied to the separation of kaempferol and other flavonoids from complex systems.The kaempferol-MIPs with high affinity and selectivity could be prepared by using kaempferol as template molecule,4-vinylpyridine as functional monomer,ethylene glycol dimethacrylate or divinylbenzene as crosslinking agent,and acetone or acetonitrile as solvent.Scanning electron microscopy and Brunauer-Emmett-Teller measurements showed that the prepared molecularly imprinted polymers had heterogeneous pore structure and large specific surface area.The properties of molecularly imprinted polymers were evaluated by static and dynamic adsorption experiments and solid phase extraction（SPE）experiments.Only the MIPs with both higher distribution coefficient and imprinting factor in the dynamic experiments had better ability to retain kaempferol.Using the screened MIPs as SPE sorbents,kaempferol and quercetin were extracted directly from the crude Ginkgo leaves hydrolysate with high recovery and purity.Control experiments also showed that the screened MIPs were superior to the commercially available adsorbents in terms of the separation of kaempferol from crude extracts.（2）Using nitric acid as catalyst,Polyethylene glycol（PEG）as pore-inducing agent and ethyl orthosilicate（TEOS）as silicon source,monolithic silica columns with homogeneous pore structure,high permeability and high porosity can be successfully prepared by sol-gel method.The effects of the content of nitric acid,the type and content of PEG and the content of TEOS on the morphology and structure of the monolithic silica column were studied in detail.The results showed that the homogeneous pore structure of silica monolith could be formed only when the concentrations of nitric acid,PEG and TEOS were at suitable levels.The PEG molecular weight had an effect on the size and distribution range of pore size.Ammonia treatment was beneficial to the stability of pore structure and skeleton of silica monolith.Electron microscope scanning observation and pore size analysis showed that the silica monolith had a large number of macroporous and mesoporous structures.The porosity of the silica monolith in methanol was as high as 88%,and the permeability coefficient was 6.67×10^-6m²,indicating that the silica monolith has good permeability.（3）After amino functionalization of the walls of the silica monoliths,kaempferol molecularly imprinted monolithic columns were easily prepared by grafting kaempferol molecularly imprinted polymers onto the walls of silica monoliths via thermal polymerization.The polymer content of the imprinted monolithic column was measured as26.5%.Scanning electron microscopy observation and pore size analysis showed that the monolithic column had homogeneous pore structure and contained abundant macropores and mesopores.The porosity was 74%.A 4.6×20 mm monolithic column was connected to a high performance liquid chromatography（HPLC）with methanol as the mobile phase,and the permeability of the column was measured as 5.78×10^-6m².It was used for chromatographic analysis of genistein,kaempferol,isorhamnetin and quercetin mixed samples.The results showed that it had good separation effect,and the separation degrees of two adjacent substances were 2.6,0.99 and 1.82,respectively.The linear determination coefficients R²of genistein,kaempferol,isorhamnetin and quercetin were 0.997,0.999,0.998 and 0.968,respectively.The LOD were 0.51,0.21,0.50 and 12.25 mg·L^-1,respectively.The LOQ were 1.01,0.63,1.00 and 24.5 mg·L^-1,respectively.The recoveries were 87.55-87.73%,74.24-103.32%,78.18-78.94%,99.18-122.52%,respectively.The linear range of kaempferol was 1-900 mg·L^-1,the relative standard deviation（RSD）of intra-day and inter-day precision were 1.9%and 2.3%respectively.For the separation and analysis of flavonoids in the complex system of Ginkgo biloba leaves,almost all impurities were eluted before 5 minutes,and two chromatographic peaks for kaempferol and isorhamnetin were finally appeared clearly.These results indicate that the molecularly imprinted monolithic column can be effectively used for the separation and analysis of flavonoids with similar structure and flavonoids from Ginkgo biloba leaf extract.In conclusion,this study is of great significance for improving the separation and analysis efficiency of flavonoids in complex systems.At the same time,the construction of molecularly imprinted monolithic columns by surface imprinting method will further expand the application of molecularly imprinted monolithic columns in the separation and analysis of natural products.