HPLC测定当归中的阿魏酸(Ferulic acid)

1)试剂

   a. 重蒸水  b. 甲醇（色谱纯）   c.乙酸（分析纯）   

2)仪器

   高效液相色谱仪（Waters）                 分析天平（1/10000）

   超声波清洗仪                            0.45mm微孔过滤器

   50ml容量瓶                              带塞小玻璃瓶

3)色谱条件

   色谱柱  SymmetryR  C18  5mm    3.9×150mm

   波长     320nm

   流动相   A: 2%乙酸  B: 甲醇

        A:B=65:35(等梯度)

   进样量   10ml

   流速     1.0ml/min

   柱温:     30℃

4)样品的制备

（1）待测样品：精确称取60mg左右提取物于25ml容量瓶中，用甲醇：乙酸（V/V=95:5）溶解，超声定容，0.45mm微孔滤膜过滤即为供试液。

（2）标准样品:  精确称取阿魏酸标准品用甲醇:乙酸(V/V=95:5)溶解,使其浓度各为0.2mg/ml, 0.45mm微孔滤膜过滤。

5)计算

根据各峰峰面积利用外标法计算各种成份的含量。

 Determination  of  Ferulic acid  by  HPLC

1) Reagents

a. Nanopure water    b. Methanol (HPLC)          c.Acetic acid

2) Apparatus

HPLC (Waters)             Analytical balance (1/10000)           Ultrasonic cleaner

0.45μm PTFE filter         50ml volumetric flask                 Screw cap vials

3) HPLC condition

Column:  SymmetryR C₁₈ 5μm , 3.9×150mm

Detector:  UV 320nm

Mobile Phase: A=2%Acetic acid    B= Methanol

A/B=65/35(isocratic)      

Flow Rate: 1.0ml/min

Injection: 10μl

4) Procedure

Samples Preparation

  Accurately weigh 60mg extraction into 25ml volumetric flask. Add Methanol :Acetic acid(V/V=95:5) to volume and sonicate to mix, filter through a 0.45μm PTFE filter into a screw cap vial and proceed with the analysis.

Standard preparation /calibration

Prepare a standard solution (approximately 0.2mg/ml in water) using a commercial reference standard of Ferulic acid，Sonicate for about 10 minutes and transfer to screw-cap vials and can be stored in the refrigeration.

5) Calculation

Determine the peak areas of the Ferulic acid on the sample chromatogram and compare to the corresponding peaks in the chromatogram for the reference standard solution. Calculate the weight percent.