類黃酮分子印跡聚合物的合成及其在柑橘中識(shí)別性能的研究
發(fā)布時(shí)間：2018-08-20 08:31
【摘要】：分子印跡技術(shù)(Molecularly imprinted technique,MIT)是制備對(duì)目標(biāo)分子有特異性識(shí)別能力的分子印跡聚合物(Molecularly imprinted polymer,MIP)的實(shí)驗(yàn)室技術(shù)。分子印跡聚合物具有高特異性、高選擇性、高強(qiáng)度等特點(diǎn),而且制備過(guò)程簡(jiǎn)單、成本低、可重復(fù)利用、耐受高溫、高壓、酸堿和有機(jī)溶劑。目前主要應(yīng)用于目標(biāo)分子的分離識(shí)別、對(duì)映體的分離、臨床藥物分析、催化劑和吸附劑等方面。已在生物工程、食品工業(yè)、臨床醫(yī)學(xué)、環(huán)境監(jiān)測(cè)等行業(yè)形成產(chǎn)業(yè)化的應(yīng)用,尤其分子印跡固相萃取(MISPE)技術(shù)在食品營(yíng)養(yǎng)功能成分的分離、富集和純化等方面有廣泛的應(yīng)用。本研究以柑橘中具有營(yíng)養(yǎng)價(jià)值的類黃酮(FLDS)為研究對(duì)象,進(jìn)行了類黃酮分子印跡聚合物的制備以及一系列相關(guān)的研究。本文綜述了柑橘中類黃酮的分離富集技術(shù)、分子印跡聚合物的制備、分子印跡聚合物的表征以及分子印跡固相萃取技術(shù)。本研究利用分子印跡技術(shù)的原理,以單一類黃酮為模板分子,優(yōu)化了制備條件,得到具有類特異性吸附、高選擇性的分子印跡聚合物微球,采用紫外光譜(UV)、傅里葉紅外光譜(FTIR)、掃描電鏡(SEM)等手段對(duì)聚合物進(jìn)行表征。用聚合物微球做填充材料制備了分子印跡固相萃取柱,利用其類特異性吸附的特點(diǎn)來(lái)分離和富集結(jié)構(gòu)類似的一類物質(zhì),實(shí)現(xiàn)了柑橘?gòu)?fù)雜基質(zhì)中類黃酮的高效分離與凈化。本研究的主要成果和結(jié)論:1.采用分子印跡技術(shù),以橙皮素(HET)為模板分子,2-乙烯基吡啶(2-VP)為功能單體,乙二醇二甲基丙烯酸酯(EGDMA)為交聯(lián)劑,乙腈為致孔劑,沉淀聚合法制備了具有類特異性識(shí)別能力的橙皮素分子印跡聚合物。用紫外光譜確定了功能單體的種類、模板分子與功能單體的配比。分別測(cè)定了不同交聯(lián)劑用量和致孔劑用量下制備的印跡聚合物的靜態(tài)吸附性能,以此確定了交聯(lián)劑用量和致孔劑的用量。實(shí)驗(yàn)結(jié)果表明,當(dāng)模板分子、功能單體、交聯(lián)劑的比例為1:6:20時(shí),致孔劑乙腈用量為50 m L時(shí),得到的分子印跡聚合物具有很好的吸附性能。在此基礎(chǔ)上用傅里葉紅外(FTIR)、掃描電鏡(SEM)等手段對(duì)分子印跡聚合物進(jìn)行表征。通過(guò)靜態(tài)吸附性能和Scatchard分析,研究了分子印跡聚合物的平衡吸附性能,試驗(yàn)結(jié)果表明MIP有兩類不同類型的結(jié)合位點(diǎn):特異性結(jié)合位點(diǎn)和非特異性結(jié)合位點(diǎn)。MIP所對(duì)應(yīng)的K_(d1)=0.243μmol/m L,Q_(max1)=143.61μmol/g;K_(d2)=3.88μmol/m L,Q_(max2)=5.29μmol/g。而NIP線性關(guān)系較好,說(shuō)明NIP只有一種非特異性結(jié)合位點(diǎn),其所對(duì)應(yīng)的K_d=0.58μmol/m L,Q_(max)=30.86μmol/g。2.實(shí)驗(yàn)評(píng)價(jià)了橙皮素分子印跡聚合物的選擇性,實(shí)驗(yàn)結(jié)果表明印跡聚合物對(duì)四種黃烷酮印跡因子分別為1.40,1.39,1.59和2.89。以橙皮素分子印跡聚合物為填充料制備了分子印跡固相萃取小柱,篩選和優(yōu)化了固相萃取條件,建立了基于MISPE技術(shù)快速分離和富集四種黃烷酮(FLES)的高效液相色譜方法,研究了分子印跡聚合物固相萃取柱的實(shí)用性,實(shí)現(xiàn)了枳實(shí)中柚皮苷(NGIN)、橙皮苷(HES)、柚皮素(NGEN)、橙皮素(HET)的分離和富集,該方法在2~120 mg/L濃度范圍內(nèi)具有良好的線性關(guān)系(R~2≥0.9991),檢出限(LOD)范圍0.2~0.5 mg/L。分子印跡固相萃取柱(MISPE)對(duì)四種黃烷酮的提取率大小順序?yàn)?NGENHETNGINHES,檢測(cè)出枳實(shí)中四種黃烷酮的含量分別為0.075 mg/g(NGIN),7.55 mg/g(HES),0.49mg/g(NGEN),0.29 mg/g(HET),說(shuō)明MIP對(duì)四種黃烷酮有較好的識(shí)別能力和類特異性吸附性能,可以很好地應(yīng)用于枳實(shí)中四種黃烷酮的分離與富集。3.以桔皮素為模板分子,丙烯酰胺(AM)為功能單體,乙二醇二甲基丙烯酸酯(EGDMA)為交聯(lián)劑,乙腈為致孔劑,沉淀聚合法制備了桔皮素分子印跡聚合物,并優(yōu)化了合成條件。采用傅里葉紅外光譜(FTIR)和掃描電鏡(SEM)等手段對(duì)分子印跡聚合物進(jìn)行表征。通過(guò)靜態(tài)吸附性能實(shí)驗(yàn)來(lái)測(cè)定聚合物對(duì)三種多甲氧基黃酮的吸附性能,結(jié)果表明MIP對(duì)甜橙黃酮(SNT)、川皮苷(NBT)和桔皮素(TNG)具有較好的吸附性能,最大吸附量分別為6.67μmol/g,6.34μmol/g,7.19μmol/g。以傘形花內(nèi)酯(7-HC)為參照,評(píng)價(jià)了MIP對(duì)三種多甲氧基黃酮(PMFS)的選擇性,結(jié)果顯示MIP對(duì)甜橙黃酮、川皮苷、桔皮素和傘形花內(nèi)酯的印跡因子IF分別為1.56、1.83、1.46、0.96。將印跡聚合物微球作為色譜填料制備分子印跡固相萃取柱,用于分離和富集柑橘果皮中三種多甲氧基黃酮,評(píng)價(jià)了分子印跡固相柱的實(shí)用性和選擇性。建立了甜橙黃酮、川皮苷、桔皮素的分子印跡聚合物-高效液相色譜檢測(cè)方法,實(shí)現(xiàn)了柑橘中三種多甲氧基黃酮的快速分離和高效富集,該方法在2~120 mg/L濃度范圍內(nèi)具有良好的線性關(guān)系,相關(guān)系數(shù)(R~2)皆為0.9999,檢出限為0.2~0.5 mg/L,RSD≤1.9%。該固相萃取柱對(duì)三種多甲氧基黃酮檢測(cè)含量分別為0.49 mg/g(SNT),1.13 mg/g(NBT),0.82 mg/g(TNG),說(shuō)明該固相萃取柱對(duì)三種多甲氧基黃酮具有較好的識(shí)別能力和選擇性,分離效果良好,有效去除了樣品中雜質(zhì)的干擾,大大提高了柑橘果皮中的三種多甲氧基黃酮的萃取效率。
[Abstract]:Molecularly imprinted technology (MIT) is a laboratory technology for the preparation of molecularly imprinted polymer (MIP) with specific recognition ability to target molecules. Utilization, tolerance to high temperatures, high pressures, acids, bases and organic solvents. Currently, it is mainly used in the separation and identification of target molecules, enantiomer separation, clinical drug analysis, catalysts and adsorbents. It has been industrialized in bioengineering, food industry, clinical medicine, environmental monitoring and other industries, especially molecular imprinting solid phase extraction (MISPE) technology. Atractylodes molecularly imprinted polymers of flavonoids (FLDS) with nutritional value in citrus were prepared and a series of related studies were carried out. The separation and enrichment techniques and molecular imprinting of flavonoids in citrus were reviewed in this paper. The preparation of imprinted polymers, characterization of molecularly imprinted polymers and molecular imprinting solid phase extraction (MISPE) were studied. Based on the principle of molecularly imprinted technology, single flavonoid was used as template molecule to optimize the preparation conditions. Molecularly imprinted polymer microspheres with class-specific adsorption and high selectivity were prepared. Molecularly imprinted solid phase extraction column was prepared by using polymer microspheres as filling materials. A class of substances with similar structure were separated and enriched by using their specific adsorption characteristics. The high efficiency separation and purification of flavonoids in Citrus complex matrix was achieved. The main results and conclusions are as follows: 1. Hesperidin-like molecular imprinted polymers with specific recognition ability were prepared by molecular imprinting technique, using hesperidin (HET) as template molecule, 2-vinylpyridine (2-VP) as functional monomer, ethylene glycol dimethacrylate (EGDMA) as crosslinking agent and acetonitrile as porogen. The types of functional monomers and the ratio of template molecules to functional monomers were determined. The static adsorption properties of the imprinted polymers were measured under different dosages of cross-linking agents and porogens, and the dosages of cross-linking agents and porogens were determined. Molecularly imprinted polymers with 50 m L acetonitrile were characterized by Fourier transform infrared spectroscopy (FTIR) and scanning electron microscopy (SEM). The equilibrium adsorption properties of the molecularly imprinted polymers were studied by static adsorption and Catchard analysis. The experimental results were presented. MIP has two different types of binding sites: specific binding sites and nonspecific binding sites. The corresponding K_ (d1) = 0.243 micromol/m L, Q_ (max1) = 143.61 micromol/g, K_ (d2) = 3.88 micromol/m L, Q_ (max2) = 5.29 micromol/g. However, NIP has a good linear relationship, indicating that there is only one non-specific binding site, and the corresponding K_d = 0.58 micromol/m, Q_ (d2) = 3.88 micromol/g. _ (max) = 30.86 micromol / g.2. The selectivity of hesperidin molecularly imprinted polymers was evaluated. The results showed that the imprinted polymers had 1.40, 1.39, 1.59 and 2.89 flavanone imprinting factors, respectively. A high performance liquid chromatography (HPLC) method based on MISPE for rapid separation and enrichment of four flavanones (FLES) was established. The practicability of molecularly imprinted polymer solid phase extraction column was studied. The separation and enrichment of naringin (NGIN), hesperidin (HES), naringin (NGEN) and hesperidin (HET) in Fructus Aurantii Immaturus were realized. The method had good performance in the range of 2-120 mg/L. The linear relationship (R~2 < 0.9991) and the detection limit (LOD) ranged from 0.2 mg/L to 0.5 mg/L. The order of extraction rates of four flavanones from Fructus Aurantii by molecularly imprinted solid phase extraction column (MISPE) was NGENHE TNGINHES. The contents of four flavanones in Fructus Aurantii were 0.075 mg/g (NGIN), 7.55 mg/g (HES), 0.49 mg/g (NGEN) and 0.29 mg/g (HET), respectively. Hesperidin molecularly imprinted polycondensation was prepared by precipitation polymerization using hesperidin as template molecule, acrylamide (AM) as functional monomer, ethylene glycol Dimethylacrylate (EGDMA) as crosslinking agent and acetonitrile as porogen. The molecularly imprinted polymers were characterized by Fourier transform infrared spectroscopy (FTIR) and scanning electron microscopy (SEM). The adsorption properties of the polymers for three polymethoxy flavonoids were determined by static adsorption experiments. The results showed that MIP had better adsorption properties for SNT, NBT and TNG. The maximum adsorption capacity of MIP was 6.67, 6.34 and 7.19 micromol/g, respectively. The selectivity of MIP to three polymethoxy flavonoids (PMFS) was evaluated with umbelliferol (7-HC). The results showed that the imprinting factors IF of MIP to orange flavonoids, kaempferide, hesperidin and umbelliferolide were 1.56, 1.83, 1.46, 0.96, respectively. Compound microspheres were used as chromatographic fillers to prepare molecularly imprinted solid phase extraction column for separation and enrichment of three polymethoxy flavonoids from citrus peel. The practicability and selectivity of molecularly imprinted solid phase column were evaluated. Rapid separation and high-efficiency enrichment of polymethoxy flavonoids showed that the method had a good linear relationship in the range of 2-120 mg/L. The correlation coefficients (R~2) were 0.9999, the detection limits were 0.2-0.5 mg/L and RSD was less than 1.9%. The detection contents of three polymethoxy flavonoids were 0.49 mg/g (SNT), 1.13 mg/g (NBT) and 0.82 mg/g (TNG), respectively. The results showed that the solid phase extraction column had good recognition ability and selectivity for three polymethoxy flavonoids, and the separation effect was good. The interference of impurities in the sample was effectively removed, and the extraction efficiency of three polymethoxy flavonoids in citrus peel was greatly improved.
【學(xué)位授予單位】：西南大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：O631.3;TS255.1
本文編號(hào)：2193023
[Abstract]:Molecularly imprinted technology (MIT) is a laboratory technology for the preparation of molecularly imprinted polymer (MIP) with specific recognition ability to target molecules. Utilization, tolerance to high temperatures, high pressures, acids, bases and organic solvents. Currently, it is mainly used in the separation and identification of target molecules, enantiomer separation, clinical drug analysis, catalysts and adsorbents. It has been industrialized in bioengineering, food industry, clinical medicine, environmental monitoring and other industries, especially molecular imprinting solid phase extraction (MISPE) technology. Atractylodes molecularly imprinted polymers of flavonoids (FLDS) with nutritional value in citrus were prepared and a series of related studies were carried out. The separation and enrichment techniques and molecular imprinting of flavonoids in citrus were reviewed in this paper. The preparation of imprinted polymers, characterization of molecularly imprinted polymers and molecular imprinting solid phase extraction (MISPE) were studied. Based on the principle of molecularly imprinted technology, single flavonoid was used as template molecule to optimize the preparation conditions. Molecularly imprinted polymer microspheres with class-specific adsorption and high selectivity were prepared. Molecularly imprinted solid phase extraction column was prepared by using polymer microspheres as filling materials. A class of substances with similar structure were separated and enriched by using their specific adsorption characteristics. The high efficiency separation and purification of flavonoids in Citrus complex matrix was achieved. The main results and conclusions are as follows: 1. Hesperidin-like molecular imprinted polymers with specific recognition ability were prepared by molecular imprinting technique, using hesperidin (HET) as template molecule, 2-vinylpyridine (2-VP) as functional monomer, ethylene glycol dimethacrylate (EGDMA) as crosslinking agent and acetonitrile as porogen. The types of functional monomers and the ratio of template molecules to functional monomers were determined. The static adsorption properties of the imprinted polymers were measured under different dosages of cross-linking agents and porogens, and the dosages of cross-linking agents and porogens were determined. Molecularly imprinted polymers with 50 m L acetonitrile were characterized by Fourier transform infrared spectroscopy (FTIR) and scanning electron microscopy (SEM). The equilibrium adsorption properties of the molecularly imprinted polymers were studied by static adsorption and Catchard analysis. The experimental results were presented. MIP has two different types of binding sites: specific binding sites and nonspecific binding sites. The corresponding K_ (d1) = 0.243 micromol/m L, Q_ (max1) = 143.61 micromol/g, K_ (d2) = 3.88 micromol/m L, Q_ (max2) = 5.29 micromol/g. However, NIP has a good linear relationship, indicating that there is only one non-specific binding site, and the corresponding K_d = 0.58 micromol/m, Q_ (d2) = 3.88 micromol/g. _ (max) = 30.86 micromol / g.2. The selectivity of hesperidin molecularly imprinted polymers was evaluated. The results showed that the imprinted polymers had 1.40, 1.39, 1.59 and 2.89 flavanone imprinting factors, respectively. A high performance liquid chromatography (HPLC) method based on MISPE for rapid separation and enrichment of four flavanones (FLES) was established. The practicability of molecularly imprinted polymer solid phase extraction column was studied. The separation and enrichment of naringin (NGIN), hesperidin (HES), naringin (NGEN) and hesperidin (HET) in Fructus Aurantii Immaturus were realized. The method had good performance in the range of 2-120 mg/L. The linear relationship (R~2 < 0.9991) and the detection limit (LOD) ranged from 0.2 mg/L to 0.5 mg/L. The order of extraction rates of four flavanones from Fructus Aurantii by molecularly imprinted solid phase extraction column (MISPE) was NGENHE TNGINHES. The contents of four flavanones in Fructus Aurantii were 0.075 mg/g (NGIN), 7.55 mg/g (HES), 0.49 mg/g (NGEN) and 0.29 mg/g (HET), respectively. Hesperidin molecularly imprinted polycondensation was prepared by precipitation polymerization using hesperidin as template molecule, acrylamide (AM) as functional monomer, ethylene glycol Dimethylacrylate (EGDMA) as crosslinking agent and acetonitrile as porogen. The molecularly imprinted polymers were characterized by Fourier transform infrared spectroscopy (FTIR) and scanning electron microscopy (SEM). The adsorption properties of the polymers for three polymethoxy flavonoids were determined by static adsorption experiments. The results showed that MIP had better adsorption properties for SNT, NBT and TNG. The maximum adsorption capacity of MIP was 6.67, 6.34 and 7.19 micromol/g, respectively. The selectivity of MIP to three polymethoxy flavonoids (PMFS) was evaluated with umbelliferol (7-HC). The results showed that the imprinting factors IF of MIP to orange flavonoids, kaempferide, hesperidin and umbelliferolide were 1.56, 1.83, 1.46, 0.96, respectively. Compound microspheres were used as chromatographic fillers to prepare molecularly imprinted solid phase extraction column for separation and enrichment of three polymethoxy flavonoids from citrus peel. The practicability and selectivity of molecularly imprinted solid phase column were evaluated. Rapid separation and high-efficiency enrichment of polymethoxy flavonoids showed that the method had a good linear relationship in the range of 2-120 mg/L. The correlation coefficients (R~2) were 0.9999, the detection limits were 0.2-0.5 mg/L and RSD was less than 1.9%. The detection contents of three polymethoxy flavonoids were 0.49 mg/g (SNT), 1.13 mg/g (NBT) and 0.82 mg/g (TNG), respectively. The results showed that the solid phase extraction column had good recognition ability and selectivity for three polymethoxy flavonoids, and the separation effect was good. The interference of impurities in the sample was effectively removed, and the extraction efficiency of three polymethoxy flavonoids in citrus peel was greatly improved.
【學(xué)位授予單位】：西南大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：O631.3;TS255.1
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2 吳桂蘋;柑桔果實(shí)主要類黃酮成分檢測(cè)及含量分析[D];西南大學(xué);2007年
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