中药分析实验1

实验一比色法测定大黄中有效成分的总量学时分配：6学时实验目的和要求1.掌握羧基蒽醌苷类及游离羧基蒽醌的性质；2.通过本次实验了解大黄类药材中有效成分的含量测定方法。

实验原理有色物质的溶液对不同波长的入射光线有不同程度的吸收，这是物质的性质。

溶液颜色的深浅往往是溶液浓度高低的标志，而颜色的深浅与对一定波长的吸收度有关。

因此，可以通过比较溶液对被吸收光的吸收度来测定溶液中物质的含量。

实验条件1.仪器：分光光度计，移液管，容量瓶，锥形瓶，量筒。

2.试剂：NaHCO3, 10%FeCl3, 浓盐酸，氯仿，0.5% 醋酸镁甲醇溶液。

实验内容1.对照品溶液及标准曲线：精密称取1，8 –二羟基蒽醌2.5 mg 于25 ml 容量瓶中，加氯仿溶解并加至刻度，精密吸取对照品溶液0.5，1，2，3，4 ml ，分别置于25 ml 容量瓶中，蒸去氯仿，加0.5% 醋酸镁甲醇溶液至刻度，摇匀。

以甲醇为空白，在515 nm 处测定，以吸收度为纵坐标，浓度(μg / ml)为横坐标，绘制标准曲线。

2.定量分析精密称取药材粉0.1 g ，置100 ml 烧瓶中，精密加入水25 ml ，称定重量。

直火加热15 分钟，放冷，称定重量，用水补足至原重量，加碳酸氢钠50 mg ，摇匀，静置后用干燥滤纸滤过，弃去初滤液，精密吸取续滤液10 ml ，置100 ml 具塞烧瓶中，加10%FeCl3溶液20 ml ，回流20 分钟，加HCl 1ml ，继续回流30 分钟，放冷，加入氯仿25 ml ，盖好瓶盖，用力振摇，转移至125 ml 分液漏斗中，使分层，分取氯仿层，再用20 ml 氯仿（洗涤烧瓶后）萃取一次，分取氯仿层与上次合并，并用水洗涤二次，每次用水15 ml ，将氯仿层用干燥滤纸滤过于50 ml 容量瓶中，用氯仿洗涤滤纸，洗液合并于容量瓶中，用氯仿稀释至刻度，摇匀。

精密吸取5 ml 氯仿提取液，置25 ml 具塞烧瓶中，蒸去氯仿，放冷，精密加入0.5% 醋酸镁甲醇溶液10 ml 。

摇匀，以甲醇为空白，在515 nm 处测定吸收值，由标准曲线计算含量。

实验记录1.绘标准曲线图。

2.计算大黄中结合蒽醌和游离大黄酸的总含量。

思考题1.加水提取的是何类成分,加入10%FeCl3的目的是什么?2.加入浓盐酸的目的是什么?3.用比色法测定蒽醌类成分应注意什么?Experiment 1 Determination of the Total Content of Effective Componentsin Radix et Rhizoma Rhei by Colorimetric Method Class hour: 6Objectives1.Master the properties of carboxylic anthraquinone glycosides and free carboxylicanthraquinones.prehand the assay method of effective components in Radix et Rhizoma Rheithrough the experiment.PrincipleSolution of colored substance can absorb beam of different wavelength in different degree, which is one of the properties of substance. The solution’s color always indicates the concentration, while the color is relevant to the absorbance at a definite wavelength. Thus, we can determinate the content of substance in solution by the means of comparing the absorbance of the light absorbed by the solution. Conditions1. Apparatus: spectrophotometer, pipette, volumetric flask, conical flask, graduated cylinder.2. Reagents: NaHCO3, 10%FeCl3, concentrated hydrochloric acid, chloroform, 0.5% magnesium actate in methanol.Procedure1. Preparation of reference solution and standard curve:2.5mg of 1, 8-dihydroxy-anthraquinone is accurately weighed into a 25mL volumetric flask, dissolved in chloroform and diluted to the volume. Transfer 0.5, 1, 2, 3, 4mL of the reference solution accurately into 25mL volumetric flasks respectively. After evaporating the chloroform, dilute the solution with 0.5% magnesium acetate in methanol to the volume, and mix it well. Methanol is used as the blank control. We can measure the absorbance at 515 nm and prepare the standard curve with the absorbance as ordinate and the concentration (µg/mL) as abscissa.2. Quantitative analysis:Accurately 0.1g of the powder is weighed into a 100 mL conical flask andaccurately 25mL water is added, then the flask is weighed and heated under reflux on a direct fire for 15mins. The loss of the water is replenished after the flask is cool and 50 mg of sodium bicarbonate is added. Then, this solution is mixed well, allowed it to stand, and filtered through a piece of dry filter paper. The initial filtrate is discarded, accurately 10mL of the successive filtrate is transferred to a 100mL conical flask, and 20mL of 10% ferric chloride solution is added. This flask is heated under reflux on a direct fire again for 20 mins, and 1mL of hydrochloric acid is added, that is continue to be heated under reflux for 30 mins more. At last, 20mL of chloroform is added after the flask is cool, which is vigorously shaken with tightly stoppered. The mixture solution is transferred to a separating funnel, which is allowed to stand. After the chloroform layer is separated and 20mL of chloroform are added to wash the conical flask and transfer it to the separating funnel. The combined chloroform extracts is washed with two 15mL quantities of water. This chloroform solution is filtered through a piece of dry filter paper to a 50mL volumetric flask, and the filter paper is washed with chloroform to the same flask. Some chloroform is added to volume and the solution is mixed well. Then, accurately 5 mL of the chloroform solution is transferred to a 25mL stopper conical flask and the chloroform is evaporated. Accurately 10mL of 0.5% solution of magnesium acetate in methanol is added after this conical flask is cool. The absorbance at 515nm is measured by using the methanol as the blank, and the content of anthraquinone is calculated by the stand curve.Experiment records1. Plot the standard curve of reference solution.2. Calculate the total content of combined anthraquinone and free rhein in the Radix et Rhizoma Rhei (Dahuang)Questions1.Which kind of component was extrac ted by water? What’s the use of 10%FeCl3?2.What’s the use of concentrated hydrochloric acid? Which procedure we should pay more attention to in this experiment?。