Determination of Chlorogenic acid by HPLC 1) Reagents a. Methanol(HPLC) b. Nanopure water c.Alcohol (A.R) d.Phosphoric acid(A.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: Kromasil C18 5m 4.6×200mm Detector: UV 324nm Mobile Phase: A=Water(91%) B=Methanol(9%) Flow Rate: 1.3 ml/min Injection: 10μl
4) Procedure Samples Preparation Accurately weigh 60mg extraction into 50ml volumetric flask. Add Alcohol:water=7:3(v/v) 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 0.1mg/ml in Alcohol:water=7:3 ) using a standard of Chlorogenic 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 Chlorogenic acid 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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