|Determination of Citrus Polyphenols by HPLC
a. Methanol (HPLC) b. Nanopure water
c. Phosphate acid(A.R)
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=0.1% Phosphate acid B= MeOH
Time（min） 0.01 35 38 40 45
%A 69 28 28 69 stop
%B 31 72 72 31
Accurately weigh 30mg extraction into 50ml volumetric flask. Add MeOH 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 0.6mg/ml in water) using a commercial reference of Citrus Polyphenols，Sonicate for about 10 minutes and transfer to screw-cap vials and can be stored in the refrigeration.
Compare the peak areas of the Citrus Polyphenols on the sample chromatogram and the corresponding peaks in the chromatogram for the reference solution.