紫外吸收光度法鉴别和测定维生素B12注射液(双语)
紫外吸收光度法鉴别和测定维生素B 12注射液
【目的要求】
1．掌握紫外分光光度计的基本结构及使用方法；
2．掌握维生素B 12注射液的鉴别和含量测定方法。
【实验原理】
1. 紫外分光光度计的组成与结构
紫外分光光度计主要由光源（D 灯、W 灯）、单色器（棱镜、光栅、滤光片）、吸收池、检测器（光电管、光电倍增管、光电池）以及信号放大处理和显示系统等部分组成，其基本结构和工作流程如同21-1所示。
图21-1 单光束紫外分光光度计的基本结构
2. 维生素B 12的定性鉴别
由于物质的性质和结构不同，物质对光的吸收是具有选择性的。维生素B 12的吸收曲线反映了其化学结构的基本特征，分别在278nm 、361nm 和550nm 波长处有三个吸收峰，因此可通过它的吸收光谱的特征，利用其特征吸收值之间的比值A 361/A 278和A 361/A 550进行定
性鉴别，并了解维生素B 12的纯度，
药典规定：A 361/A 278和A 361/A 550的比值应分别在1.70~1.88和3.15~3.45之间。
3. 维生素B 12注射液的定量分析
由于维生素B 12在361nm 处的溶液吸收最强（2071%
1cm,361nm =E ），且干扰因素少，因此以361 nm 作为测量波长，在此波长下测量其溶液的吸光度A ，采用吸光系数法直接测定浓度，按下式计算维生素B 12的百分含量：
由 Cl E A 1%1cm = 则 g/ml)( 31.4810207
41%1cm μA A l E A
C ×=×== %1005
.031.48(%)VitB 12×××=
∴n A （n 为稀释倍数） 【仪器与试剂】
１. 仪器
光闸
TU1810紫外-可见分光光度计，1 cm石英比色皿。
２. 试剂
维生素B12对照品，维生素B12注射液（标示量0.5 mg/ml）。
【实验内容】
1. 对照品溶液和样品溶液的配制
准确称取维生素B12对照品适量，加水溶解稀释成25μg/mL的溶液。
精密吸取维生素B12注射液0.5 ml于10 ml容量瓶中，用水稀释至刻度摇匀，得到待测样品溶液（按标示含量稀释成25μg/ml的溶液）。
2. 吸收光谱的绘制与注射液的鉴别
取对照品溶液进行光谱扫描（250 ~ 600 nm），得到维生素B12的吸收曲线，寻找吸收峰，扫描后可得到在278nm，361 nm和550 nm 波长处出现的3个吸收峰，然后在以上3个吸收峰处测量维生素B12注射液的吸光度，并计算A361/A278和A361/A550的比值。
3. 维生素B12的定量测定
根据吸收曲线选择最大吸收波长（在361 nm或附近）作为测量波长，测量维生素B12样品溶液的A值，并计算其标示含量（在90 ~ 110 %之间视为合格）。
【注意事项】
1. 注意石英比色皿的操作使用；
2. 使用5联池时要注意比色皿在样品池中的位置，不可混淆；
3. 测定时不要打开仪器的样品池盖。
【思考题】
1．采用吸光系数法直接测定样品含量有何要求？
2．利用其他组同学的实验结果，比较同一溶液在不同仪器上测得的吸收曲线的形状、吸收峰波长以及相同浓度的吸光度有无不同，试作解释。
3．比较用吸光系数和校正曲线定量方法，你认为哪种方法好？为什么？
Identification and determination of vitamin B12
in the injection by UV spectrophotometry
Objectives
1．Grasp the basic configuration and the operation of UV-Vis spectrophotometer;
2．Grasp the method of identification and determination of vitamin B12 in the injection. Principles
1. The basic configuration of UV-Vis spectrophotometer
UV spectrophotometer is mainly composed of light sources, monochromator, sample cell, detector and signal processor. Light sources usually employ deuterium lamp for ultraviolet and tungsten lamp for visible light, while prism and grating are most useful monochromator. Commonly, quartz and glass are used to make sample cell. Photovoltaic cell, phototube and photomultiplier tube have used as detector successively. The configuration and operational principle of single-beam spectrophotometer are shown as figure 21-1.
Shutter
Fig. 21-1 The configuration and principle of single-beam spectrophotometer
2. Identification of vitamin B12 in sample
When electromagnetic radiation passes through a substance such as solid, liquid and gas, which can selectively absorb certain frequencies of radiation because of its structure and properties. The ultraviolet absorption spectrum of vitamin B12 reveals its characteristic in chemical structure, thus three absorptive peaks appear at 278 nm, 361 nm and 361 nm of wavelengths respectively. Accordingly, based on characteristic of the spectrum, vitamin B12 can be identified by measuring the ratios of absorbances (i.e. A361/A278 and A361/A550), respectively. Furthermore, how the sample of vitamin B12 in purity can be known qualitatively. According to the values authorized by Chinese Pharmacopoeia, A361/A278 and A361/A550 in number must be between 1.70~1.88 and 3.15~3.45 respectively.
3. Determination of vitamin B12 in sample
Vitamin B12 can be determined directly by using absorption coefficient method because of
the maximum absorption (2071%1cm,361nm =E ) and less interference at 361 nm. Through measured the absorbance at the wavelength, the content of vitamin B 12 can be calculated as follows:
Cl E A 1%1cm =Q ? g/ml)( 31.4810207
41%1cm μA A l E A
C ×=×== %1005
.031.48(%)VitB 12×××=∴n A (Where n is dilution factor.) Equipment and Chemicals
Equipment:
TU1810 spctrophotometer, 1cm quartz cell;
Chemicals:
Vitamin B 12 reference and vitamin B 12 injection (500μg/ml).
Procedures
1. Preparation of standard solution and sample solution
Weigh accurately a certain quantity of vitamin B 12 reference, dissolve it with distilled water, and dilute to the concentration of 25μg/ml.
Pipet precisely 0.5 ml of the vitamin B 12 injection in 10 ml volumetric flask, dilute it to volume with distilled water and mix well (Its concentration is about 25μg/ml) .
2. Identification of vitamin B 12 in the injection
Transfer a certain volume of standard solution in quartz cell carefully, and take it in a spctrophotometer. Then scanning wavelengths are performed from 200 nm to 600 nm to draw the absorption spectrum of vitamin B 12 in a spctrophotometer. Find out the wavelengths of absorption peaks near 278 nm, 361 nm and 550 nm respectively. Next, measure the absorbances of sample solution at three wavelengths selected, and calculate the absorption ratio of A 361/A 278 and A 361/A 550 respectively.
3. Determination of vitamin B 12 in the injection
According to the spectrum of vitamin B 12, the maximum absorption wavelength near 361 nm is selected to measure the absorbance of the sample solution. Then calculate the content of vitamin B 12, which is certificated between 90 % and 110 % according to Chinese Pharmacopoeia. Notes
1. Pay attention to correct use of quartz cells;
2. Pay attention to the place of sample cells in the instrument when you measure;
3. Don’t open the cover of sample room when you measure.
Questions
1．What requirement must be met determination of content in a sample by absorption coefficient method?
2．Compare experiment results with other group of students through the shape of absorption spectra，the location of maximum absorption wavelength and the absorbance for the same solution in different instrument, and explain why they are same or different.
3．Compare absorption coefficient method with calibration curve method, Which is better? Why?