【作者】 李银花；

【导师】 刘仲华； 黄建安；

【作者基本信息】 湖南农业大学 ， 茶学， 2006， 硕士

【摘要】 茶氨酸是茶叶中特有的非蛋白质氨基酸。L-茶氨酸作为一种安全、无毒、具有多种生理功能的天然产物，具有广阔的开发应用前景。从茶叶中直接提取天然L-茶氨酸，是茶的功能成分高效利用的重要途径之一。但是，以茶叶为原料，生产高纯天然L-茶氨酸时，分离纯化成本较高，价格竞争力不强，影响了其规模化生产。而化学合成法制备茶氨酸，操作相对简单，易于工业化生产，但产品多为D-、L-的混旋体，D-茶氨酸的分离与去除具有较高的技术难度。据研究，人体需要并能被吸收利用的茶氨酸为L型。在营养学上，D-茶氨酸的生理功能与活性尚不明晰。因此，有必要对茶氨酸对映体进行定性鉴定和定量分析方法研究。本文首次系统研究了高效液相色谱法拆分和定量茶氨酸对映体的方法，同时研究了温度与L-茶氨酸异构化的关系。取得了如下具有创新性研究结果： 1．建立了配体交换色谱手性固定相法拆分茶氨酸对映体的方法。色谱条件为：采用L-羟基脯氨酸硅胶键合手性柱；流动相为0.8mmol／L的硫酸铜-水溶液；波长为254nm：流速为1.0ml／min；柱温30℃。L-茶氨酸的进样量在0.08126μg～4.514μg范围内峰面积与进样量之间线性关系良好，平均回收率为99.12％；D-茶氨酸的进样量在0.08250μg～4.583μg范围内峰面积与进样量之间线性关系良好，平均回收率为99.10％。该方法操作方便，具有良好的稳定性和准确度。 2．建立了配体交换色谱手性流动相法拆分茶氨酸对映体的方法。色谱条件为：采用Polaris C₁₈柱；流动相为L-脯氨酸：Cu²⁺（摩尔比）为2：1，Cu²⁺浓度为0.5mmol／L，pH6.8，甲醇的加入体积比为2％；波长为254nm；流速为0.9ml／min；柱温30℃。L-茶氨酸的进样量在0.09542μg～4.241μg范围内峰面积与进样量之间线性关系良好，平均回收率为98.76％；D-茶氨酸的进样量在0.08486μg～4.243μg范围峰面积与进样量之间线性关系良好，平均回收率为98.39％。该方法通用性好，准确。 3．建立了手性衍生试剂法拆分茶氨酸对映体的方法。色谱条件为：采用Kromasil C₁₈柱；流动相为三乙胺．磷酸缓冲液和乙腈，梯度洗脱；波长为340nm；流速为1.0ml／min；柱温35℃。L-茶氨酸的进样量在1.732×10^-3μg～2.077μg范围内峰面积与进样量之间线性关系良好，平均回收率为100.1％；D-茶氨酸的进样量在1.696×10^-3μg～2.044μg范围内峰面积与进样量之间线性关系良好，平均回收更多还原

【Abstract】 L-Theanine is a peculiar non-protein amino acid in tea.It is a new kind of natural product, which is safely and poisonless .L-Theanine has lots of physiological function.The future of its development is extensive. we directly extract L-theanine from tea,which is one of the important ways to use tea’s functional ingredient effectively. If we adopt tea as material to produce L-Theanine,the cost of separation and purification is high,it has’t the advantage of price,thus influence the scale of industrialization.Chemical synthesis course is simple which can be industrialized, the product is DL-enantiomer,it is difficult to separate and get rid of D-Theanine.People only need L-Theanine which can be absorable and usable.In point of nutrition,It is no perspicuity conclusion for the physiological and active function of D-Theanine .It is necessary to qualitatively and quantitatively study theanine enantiomer. The HPLC method was first established for separation and quantitation of theanine enantiomer and also studied the relationship between temperature and the transform of L-theanine.The main innovate results are as follows:l.Chiral ligand exchange chromatographic stationary phases method was established for the separation of theanin enantiomer.The optimum condition was as follows :L-Hydroxyproline bonded chiral column;mobile phase:0.8mmol/L CuSO₄-H₂O;wavelength:254nm;flow rate :1.0ml/min;column temperature:30℃. There was a good linearity between peak area and injection quantity of L-Theanine range from 0.08126μg⁴.514μg and D-Theanine range from 0.08250μg⁴.583μg.The average recovery rate was 99.12% for L-Theanine and 99.10 % for D-Theanine.The method was simple、 stable and accurate.2.Chiral ligand exchange chromatographic mobile phases method was established for the separation of theanin enantiomer.The optimum condition was as follows:Polaris C₁₈ column;mobile phase:L-proline:Cu²⁺=2:1,the concentration of Cu²⁺was 0.5mmol/L,pH6.8,the volume of methanol is 2%; wavelength:254nm;flow rate:0.9ml/min;column temperature:30℃ .There was a good linearity between peak area and injection quantity of L-Theanine range from 0.09542μg⁴.241μg and D-Theanine range from 0.08486μg⁴.243μg.The average recovery rate was 98.76%更多还原