Determination of Kavain by HPLC 1) Reagents a. Methanol (HPLC) b. Nanopure water c.Acetronitrile(HPLC) d. Phoaphate acid(C.R) 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 VP-ODS C18 5 m 4.6150mm Detector: UV 256nm Mobile Phase: A:0.1% Phoaphate acid B: MeOH: Acetronitrile=1:1 A:B=45:55 Flow Rate: 1.0ml/min Injection: 10μl
4) Procedure Samples Preparation Accurately weigh 20mg extraction into 50ml volumetric flask. Add 5% MeOH 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 mixed standard solution (approximately 0.6mg/ml in 5% MeOH) using a commercial reference standard of Kavain,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 Kavain on the sample chromatogram and compare to the corresponding peaks in the chromatogram for the reference standard solution. Calculate the weight percent.
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