Determination of Theaflavin by HPLC
1) Reagents
a. Acetronitrile(HPLC) b. Nanopure water c.Acetic acid (A.R) d.Ethyl Acetate
2) Apparatus
HPLC (Shimadzu) Analytical balance (1/10000) Ultrasonic cleaner
0.45μm PTFE filter 50ml volumetric flask Screw cap vials
3) HPLC condition
Column: Shim-pack ODS C18 5 m 4.6150mm
Detector: UV 280nm
Mobile Phase: A=2% Acetic acid B=Acetronitrile:EtoAC=21:3
Flow Rate: 0.8 ml/min
Injection: 10μl
Gradient:
Time %A %B
0.01 82 18
28 76 24
30 76 24
32 82 18
35 82 18
4) Procedure
Samples Preparation
Accurately weigh 30mg extraction into 50ml volumetric flask. Add Water 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 standard of Theaflavins，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 Theaflavins on the sample chromatogram and compare to the corresponding peaks in the chromatogram for the standard solution. Calculate the weight percent.
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