Study On Synthesis And Molecular Recognition Of Flavonoids Molecularly Imprinted Polymers In Citrus

Molecularly Imprinting technique?MIT?is a template-directed technique that allows the design and synthesis of polymers with well defined artificial generated recognition sites for a target analyte or class of analytes.MIPs are stable in a broad range of p H,pressure and temperature.Additionally,those polymeric materials are easier to synthesize for a wide range of substances and are less expensive than antibodies.These polymeric materials can be reused for several times without loss of the “memory effect”.With high selectivity and recognition,molecularly imprinted polymers were utilized to isolation of target moleculars,separation of enantiomers,analyses of clinical medicines,catalysts and sorbents etc,which widely industrial applied in biological engineering,food industry,clinical medicine,environmental monitoring.Recently,the application of MIP as selective sorbents for solid phase extraction SPE?MISPE?procedures has also emerged.They allow a simultaneous and efficient preconcentration of target analytes and clean-up of the samples,removing undesirable sample matrix component,which was popular in the separation,enrichment and purification of nutrition ingredients in food.Citrus flavonoids?FLDS?which have biological activities were selected as subjects investigated;synthesis of molecularly imprinted polymers and a series of related researches were carried out in this paper.The separation and enrichment techniques of flavonoids in C itrus,the preparation and the characterization of molecularly imprinted polymers,and the molecularly imprinted solid-phase extraction?MISPE?technique were reviewed.Based on principle of molecularly imprinted technology,the class-specific,highly selective molecularly imprinted polymer microspheres was synthesized and optimized with one kind of flavonoid as template.In the subsequent experiments,MIP was characterizied by means of UV,FTIR and SEM.As a fillig material,MIP was packed in the solid-phase extraction column to separate and enrich efffectively the flavonoids components and its analogues in citrus complex matrices.The main research achievements and conclusions were as follows:1.Hesperetin molecularly imprinted polymers?MIP?for recognizing four flavanones?naringin hesperidin,naringenin,hesperetin?were synthesized via precipitation polymerization with hesperetin?HET?as template,2-Vinylpyridine?2-VP?as functional monomer,Ethyldiol Methacrylate?EGDMA?as crosslinker and acetonitrile as porogen.The optimal functional monomer,the ratio of template and functional monomer were obtained via UV spectrum.The amount of crosslinker and porogen were obtained via static adsorption experiment.The results showed that when the ratio of template,monomer and crosslinker was 1:6:20 and the amount of acetonitrile was 50 m L,the molecularly imprinted polymers had good adsorption properties.In the subsequent experiments,the molecular imprinted polymers were characterized via Fourier transform infrared?FTIR?and scanning electron microscopy?SEM?.The equilibrium adsorption properties of molecularly imprinted polymers were investigated by static adsorption and Scatchard analysis.The results showed that two different types of binding sites of MIP were found: specific binding sites and non-specific binding sites.The dissociation constant and apparent maximum binding capacity for MIP were K_d1= 0.243 ?mol/m L,Q_max1= 143.61 ?mol/g,K_d2= 3.88 ?mol/m L and Q_max2= 5.29 ?mol/g,respectively.The NIP has a good linear relationship,which means that NIP has only one non-specific binding site.The dissociation constant and apparent maximum binding capacity for NIP were K_d= 0.58 ?mol/m L,Q_max= 30.86 ?mol/g,respectively.2.The selectivity of molecularly imprinted polymers was evaluated.The experimental results showed that the imprinted factors of the four flavanones were 1.40,1.39,1.59 and 2.89,respectively.The molecular imprinted solid extraction colomn was prepared with the hesperetin molecularly imprinted polymers as packed materials.The molecularly imprinted solid extraction conditions was optimized.Based on the molecularly imprinted solid extraction technique,the HPLC methods of fast separation and enrichment of four flavanones?naringin,hesperidin,naringenin,hesperetin?in citrus aurantium were established.To evaluate the feasibility of molecularly imprinted solid phase extraction column in crude extract solution,molecularly imprinted solid phase extraction column was applied to separate and enrich four flavanones in citrus aurantium extracts.The results show that this method has a good linear relationship in the range of 2¹20 mg/L?R²?0.9991?and the limit of detection?LOD?is 0.2⁰.5 mg/L.Extraction rate of four flavanones is: naringenin?NGEN?> hesperetin?HET?> naringin?NGIN?> hesperidin?HES?and the maximum binding capacity of four flavanones in citrus aurantium is 0.075 mg/g?NGIN?,7.55 mg/g?HES?,0.49 mg/g?NGEN?,0.29 mg/g?HET?,respectively.The results showed that MIP had good adsorption performance and class specific recognition ability of four flavanones.The molecularly imprinted solid phase extraction column can be well applied in the separation and enrichment of four flavanones in citrus aurantium.3.With tangeretin?TNG?as template molecule,acrylamide?AM?as functional monomer,ethylene glycol two methyl acrylate?EGDMA?as crosslinker and acetonitrile as porogen,the molecularly imprinted polymer was prepareed via precipitation polymerization under optimal conditions.The molecular imprinted polymers were characterized by Fourier transform infrared spectroscopy?FTIR?and scanning electron microscopy?SEM?.The adsorption properties of the molecularly imprinted polymer for three polymethoxylflavonoids were evaluated via static adsorption.The results showed that the imprinted polymer had good adsorption properties of sinensetin?SNT?,nobiletin?NBT?and tangeretin?TNG?and the maximum adsorption capacity Qmax were 6.67 ?mol/g,6.34 ?mol/g,7.19 ?mol/g,respectively.With 7-Hydroxycoumarin?7-HC?as reference standard,the adsorption property of molecularly imprinted polymer was evaluated.The study showed the molecularly imprinted polymer had a good adsorption property.The imprinting factors?IF?for SNT,NBT,TNG and 7-Hydroxycoumarin?7-HC?were 1.56,1.83,1.46,0.96,respectively.The molecularly imprinted polymer microspheres were used as chromatographic packing materials to prepare molecularly imprinted solid-phase extraction column.In order to investigate the feasibility and selectivity of molecularly imprinted solid phase column,it was utilized to separate and enrich three polymethoxyflavones in citrus peels.A molecularly imprinted solid-phase extraction-high performance liquid chromatography?MISPE-HPLC?method was established for the enrichment and determination of three polymethoxyflavones in citrus peel.This method has a good linear relationship in the range of 2¹20 mg/L,the correlation coefficient is 0.9999,the detection limit is 0.2⁰.5 mg/L,and RSD is less than 1.9%.The maximum binding capacity of three polymethoxyflavones are 0.49 mg/g?SNT?,1.13 mg/g?NBT?,0.82 mg/g?TNG?respectively.The molecularly imprinted solid phase extraction column has good recognition ability and selectivity to three polymethoxyflavones.The molecularly imprinted solid phase extraction column has good separation capability and keeps effectively away from interference of impurities in the sample.The extraction efficiency rate of three polymethoxyflavones was improved greatly.