Chlorogenic acid by HPLC

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.