|Determination of Clover by HPLC
a. Acetronitrile (HPLC) b. Nanopure water
c. Acetic acid (A.R)
HPLC (Shimadzu) Analytical balance (1/10000) Ultrasonic cleaner
0.45μm PTFE filter 50ml volumetric flask Screw cap vials
3) HPLC condition
Column: Kromasil C18 5 m 4.6200mm
Mobile Phase: A=2% Acetic acid B= Acetronitrile
Time(min) %A %B
0.01 77 23
35 65 35
50 37 63
58 37 63
58 77 23
Flow Rate: 0.8ml/min
Accurately weigh 30mg extraction into 50ml volumetric flask. Add Methanol 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 0.4mg/ml in MeOH) using a commercial reference of Clover，Sonicate for about 10 minutes and transfer to screw-cap vials and can be stored in the refrigeration.
Compare the peak areas of the Clover on the sample chromatogram and the corresponding peaks in the chromatogram for the reference solution.