蜂蜜中有机活性组分检测的色谱方法学研究

发布时间：2018-03-27 18:56

本文选题：蜂蜜　切入点：高效毛细管电泳　出处：《河南科技学院》2017年硕士论文

【摘要】：中国是世界养蜂大国,也是世界蜂蜜生产大国,另外还是蜂蜜出口量最大的国家。蜂蜜中含有多种营养物质,具有保健功能,深受人们的欢迎。蜂蜜中的很多成分都直接影响着它的颜色、香气、口感和保健功效,而其中的有机活性组分会直接影响其营养价值及其抗菌,抗氧化等生物性功能。为了更好的评价蜂蜜的品质,本论文采用色谱技术(高效液相色谱及高效毛细管电泳)建立了蜂蜜中低分子量黄酮、有机酸和水溶性维生素类化合物的毛细管电泳及高效液相色谱分离分析方法,具体结论如下:1.黄酮是蜂蜜中的重要活性成分,通过直接紫外检测毛细管区带电泳法开发了来自不同地理来源的许多蜂蜜中的类黄酮的分析方法。在25kV的分离电压下,使用20mM硼酸盐作为运行缓冲液在pH=8.4下在5min内完全分离包括芦丁、槲皮素、山奈酚和杨梅酮的四种典型黄酮类化合物。对于四种类黄酮获得了在2.0-500mg/L的范围内的良好的线性关系,并且检测限范围为1.17至1.76mg/L。四种类黄酮的回收率在80%-107%的范围内。应用该方法分离测定了中国河南省12个养蜂场蜂蜜样品中的黄酮类化合物。在许多蜂蜜样品中仅检测到芦丁和槲皮素,其可以作为鉴定来自不同地理来源的蜂蜜的标记物。2.采用毛细管电泳法,通过场放大样品注射技术(FASI)建立了7种有机酸的分析检测方法,对影响FASI的参数进行系统研究,包括pH、注射时间、水柱长度、缓冲液浓度等。在最佳条件下检测灵敏度明显增强,各组分富集倍数可增加7-72倍。酒石酸、原儿茶酸、绿原酸、草酸、没食子酸、柠檬酸和香草酸的检测限分别为0.56、0.26、0.85、0.90、0.65、0.52和0.55mg/L。该方法成功应用于来自河南省不同地市蜂场的14种蜂蜜实际样品的检测。3.采用响应曲面法,设计三因素三水平的Box-Behnken的试验,对影响毛细管电泳分离水溶性维生素B1、B3、B5、B6及维生素C的主要影响因素(缓冲溶液的浓度、分离电压和柱温)进行了研究,以五种维生素中的最大分离度和最长保留时间为响应函数,建立了相应的数学模型,得到了它们的最佳分离条件为:17mmol/L的硼砂电泳缓冲液、15 kV电压、19℃柱温,并在最优的分离条件下,3分钟内可快速分离蜂蜜样品中的五种水溶性维生素。4.建立了同时检测维生素C和多巴胺的高效液相色谱-电化学检测法。其中高效液相色谱的色谱柱采用Diamonsil C18(2)5μ150×4.6mm色谱柱,并以体积比为74:24:2的0.05mol/L磷酸二氢钠、0.02mol/L柠檬酸和甲醇的混合液为流动相,设定检测波长为280nm,流速为1mL/min。在室温条件下,维生素C的线性范围在高效液相色谱和电化学检测器中分别为2-200μmol/L和0.05-200μmol/L,检出限分别为1.68μmol/L和0.03μmol/L;多巴胺在高效液相色谱和电化学检测器中分别为2-200μmol/L和0.05-200μmol/L,检出限分别为1.25μmol/L和0.01μmol/L。高效液相色谱-电化学检测器联用的方法更为简便灵敏度和准确率更高,且选择性更好。5.建立了可靠,灵敏的反相高效液相色谱-电化学检测(HPLC-ECD)方法测定蜂蜜中三种有机酸类电化学活性化合物(没食子酸、绿原酸、咖啡酸)。色谱分离在反相C18柱上进行,流动相由4%的醋酸水溶液(pH=4.0)和甲醇组成,通过电流检测,使用工作电位为+0.80V的玻碳电极进行梯度洗脱。该方法的线性,精度,精度和定量和检测限已被验证。在优化条件下,电化学校准曲线的线性回归分析显示出良好的线性关系,在0.005 mg/L-6.0 mg/L的线性范围内,峰面积与浓度之间的回归系数超过0.99,其检测限为10-5-3×10-5 mg/L。该方法可以应用于蜂蜜中存在的三种有机酸化合物的分析。
[Abstract]:Chinese is the world beekeeping country, is also the world's honey producing countries, and is the largest in the country. The export volume of honey honey contains a variety of nutrients, with health care function, welcomed by the people. Many of the components in honey will directly affect its color, aroma, taste and health effects, and organic active components including the direct impact on the nutritional value and biological function of antibacterial, antioxidant and so on. In order to evaluate the quality of honey, this paper adopts chromatography (HPLC and HPCE) established low molecular weight honey capillary electrophoresis separation and analysis methods of flavonoids, organic acid and water soluble vitamin compounds and HPLC chromatography, specific conclusions are as follows: 1. flavone is an important active ingredient in honey, through direct UV detection by capillary zone electrophoresis was developed from different geographical origins Analysis of the flavonoids in many honey. At the separation voltage of 25kV, using 20mM borate as running buffer at pH=8.4 in 5min complete separation including rutin, quercetin, four typical flavonoids kaempferol and Yang Meitong. The good linear relationship between the four kinds of flavonoids were obtained in the range of 2.0-500mg/L the limit of detection and recovery in the range of 1.17 to four 1.76mg/L. flavonoid rate in the range of 80%-107%. The flavonoids field in honey samples of 12 beekeeping in Henan province was determined by the method of separation of Chinese. In many honey samples was detected in rutin and quercetin, which can be used as identification from different geographical origins honey marker.2. by capillary electrophoresis, through field amplified sample injection technique (FASI) was established to analyze the detection method of 7 kinds of organic acids, the effects of FASI parameters. For system research, including pH, injection time, column length, buffer concentration. Significantly enhanced the detection sensitivity in the optimum conditions, the components can be increased by 7-72 times. The enrichment of tartaric acid, protocatechuic acid, gallic acid, chlorogenic acid, oxalic acid, citric acid and vanillic acid detection limit detection of.3. were 0.56,0.26,0.85,0.90,0.65,0.52 and the 0.55mg/L. method is successfully applied to different cities of Henan province from 14 kinds of honey bee samples using response surface methodology, experimental design of three factors and three levels of Box-Behnken, the effect of capillary electrophoresis separation of water soluble vitamins B1, B3, B5, B6 and the main influence factors of vitamin C (concentration, buffer the solution of separation voltage and column temperature) were studied in five kinds of vitamins in the maximum separation degree and the long retention time of response function, the corresponding mathematical model is established, by which the optimal separation A: borax buffer solution 17mmol/L, 15 kV voltage, 19 DEG C, column temperature, and separation in the optimal condition, 3 minutes fast separation of five honey samples of water soluble vitamin.4. was developed for the determination of vitamin C and dopamine by high performance liquid chromatography with electrochemical detection. The chromatography high performance liquid chromatography using Diamonsil C18 (2) 5 ~ 150 * 4.6mm column, and the volume ratio of 74:24:2 0.05mol/L phosphate, 0.02mol/L mixture of citric acid and methanol as the mobile phase, the detection wavelength was set 280nm, the flow rate of 1mL/min. at room temperature for 2-200 mol/L and 0.05-200 respectively. Mol/L linear range of vitamin C by high performance liquid chromatography and electrochemical detector, the detection limits were 1.68 mol/L and 0.03 mol/L respectively; 2-200 mol/L and 0.05-200 mol/L dopamine in high performance liquid chromatography and electrochemical detector, detection The limits were 1.25 mol/L and 0.01 mol/L. high performance liquid chromatography with electrochemical detector is simpler and more sensitivity and higher accuracy, and better selectivity of.5. to establish a reliable and sensitive reversed-phase high-performance liquid chromatography with electrochemical detection (HPLC-ECD) determination of three organic acids in honey for electrochemically active compounds (gallic acid, chlorogenic acid, caffeic acid). Chromatographic separation was performed on a reversed-phase C18 column, the mobile phase consisted of 4% aqueous solution of acetic acid (pH=4.0) and methanol, the current detection, using the working potential of glassy carbon electrode +0.80V in linear gradient elution. The method, precision, accuracy and quantitative detection and the limit has been verified. Under the optimized conditions, the analysis showed a good linear relationship between the electrochemical linear calibration curve regression, in the linear range of 0.005 mg/L-6.0 mg/L, between the peak area and concentration of regression coefficient is more than 0.99, The detection limit of 10-5-3 x 10-5 mg/L. can be applied to the analysis of three organic acids in honey.

【学位授予单位】：河南科技学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：O657.7;S896.1

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