Determination the Proanthocyanidin of Grape Seed by HPLC
1) Reagents
a.Methanol (HPLC) b. Acetronitrile(HPLC) c. Acetic acid (A.R)
2) Apparatus
HPLC (Beckman) Analytical balance (1/10000) Ultrasonic cleaner
0.45μm PTFE filter 50ml volumetric flask Screw cap vials
3) HPLC condition
Column: Hypersil ODS C18 5μm , 4.6×200mm
Detector: UV 280nm
Mobile Phase: A=1% HAC B=1% HAC/ACN (V/V)
Gradient:
Time（min） 0 65 70 73 75
%A 95 35 10 95 stop
%B 5 65 90 5
Flow Rate: 1.0ml/min
Injection: 10μl
4) Procedure
Samples Preparation
Accurately weigh 60mg extraction into 50ml volumetric flask. Add Methanol to volume and sonicate to mix, filter through a 0.45μm PTFE filter into a screw cap vial and proceed with the analysis.
Reference preparation /calibration
Prepare an reference solution (approximately 1.0mg/ml in MeOH) using a commercial reference of Proanthocyanidin，Sonicate for about 10 minutes and transfer to screw-cap vials and can be stored in the refrigeration.
5) Calculation
Compare the peak areas of the Proanthocyanidin on the sample chromatogram and the corresponding peaks in the chromatogram for the reference solution.
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