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HPLC測定當(dāng)歸中的阿魏酸(Ferulic acid)

1)試劑

   a. 重蒸水  b. 甲醇（色譜純）   c.乙酸（分析純）   

2)儀器

   高效液相色譜儀（Waters）                 分析天平（1/10000）

   超聲波清洗儀                            0.45mm微孔過濾器

   50ml容量瓶                              帶塞小玻璃瓶

3)色譜條件

   色譜柱  SymmetryR  C18  5mm    3.9×150mm

   波長     320nm

   流動相   A: 2%乙酸  B: 甲醇

        A:B=65:35(等梯度)

   進(jìn)樣量   10ml

   流速     1.0ml/min

   柱溫:     30℃

4)樣品的制備

（1）待測樣品：精確稱取60mg左右提取物于25ml容量瓶中，用甲醇：乙酸（V/V=95:5）溶解，超聲定容，0.45mm微孔濾膜過濾即為供試液。

（2）標(biāo)準(zhǔn)樣品:  精確稱取阿魏酸標(biāo)準(zhǔn)品用甲醇:乙酸(V/V=95:5)溶解,使其濃度各為0.2mg/ml, 0.45mm微孔濾膜過濾。

5)計算

根據(jù)各峰峰面積利用外標(biāo)法計算各種成份的含量。

 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.