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Thread: How to analysis red clover extract by HPLC

  1. #1 How to analysis red clover extract by HPLC 
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    Join Date
    Aug 2006
    Determination of Daidzein ?Genistein ?Formononetin ?Bilchanin A in Red Clover P. E. by HPLC
    1. HPLC Conditions:
    Column: Inertsil ODS-3 4.6×150mm 5?m
    Mobile phase: Methanol: Acetonitrile: Water: Phosphoric acid (30: 38:
    60: 0.1, V/V)
    Detection: 262 nm
    Sensitivity: 0.01AUFS
    Flow rate: 1.0 mL/min
    Column Temperature: Room temperature
    Injection Volume: 10?l
    2. Solution Preparation
    1. Standard Preparation:
    Accurately weigh approximately 2mg of Daidzein and Genistein and Formononetin and Bilchanin A respectively into a 50ml volumetric flask. Dilute to volume with methanol.
    2. Sample Preparation:
    Accurately weigh approximately 20mg of Red Clover P. E. into a 25ml volumetric flask and add 20ml methanol. Sonicate for 30mins then to cool to room temperature. Dilute to volume with methanol. Filter it through a 0.45?m filter into an HPLC vial or centrifuge to obtain the standard
    III. Calculation
    Under above HPLC conditions, conduct the determination at stability of aparatus and baseline. The retention time of Daidzein and Genistein and Formononetin and Bilchanin A are about 4 and 6 and 9 and 18min respectively. The final results are quantified against external standard. The formula is as follow:
    Ax Ws
    X= -------------- × ------------------×S×100%
    As Wx
    X---the content of Daidzein ?Genistein ?Formononetin ?Bilchanin A
    Ax--- the peak area of sample in Daidzein ?Genistein ?Formononetin ?Bilchanin A
    As--- the peak area of standard in Daidzein ?Genistein ?Formononetin ?Bilchanin A
    Ws--- weight of standard (mg)
    Wx--- weight of sample (mg)
    S--- standard purity

    A good product is nalysised not only by HPLC, we will open more method to identify goog red clover extract continuelly.

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