Chlorogenic acid content

REAGENTS & INSTRUMENT REQUIRED:

1.   High Performance Liquid Chromatography

2.   Water HPLC grade

3. Acetronitrile HPLC grade
4. Chlorogenic acid  working standard

PROCEDURE:

Standard Preparation :

Weigh accurately about 50 mg of working standard in 25 mL volumetric flask. Add about 15 mL of methanol. Ultrasonicate for 20 minutes and make up the volume with methanol.

Sample Preparation :

Weigh accurately about 50 mg of sample of extract into 25 mL volumetric flask. Add about 15mL of methanol. Ultrasonicate for 20 minutes and make up the volume with methanol.

Ensure that the concentration of active ingredient in sample solution is approximately similar to that of standard solution and filter through 0.45μm filter paper.

For Raw material: Take 10 gm of herb powder (20#) and reflux in methanol for 1hrs twice

Chromatographic Condition:

Mobile phase :              -A - 0.1 % Trifluoroacetic acid in water, B - Acetronitrile

Column                         -Phenomenex luna 5μ, C18 (250mm x 4.6mm)

Mode                            -Gradient

                                    15% to 22.5% B in 10 minutes, then increasing to 35 % B in 5 Minute.

Injection                       -20μL

Flow rate                      -1.0 mL/minute

Detector wave length   - 280 nm

Condition                     - Room temperature

Calculation:

                                         Area of sample   X  Concentration of std    X   purity of std

  % of Ursolic acid   =- --------------------------------------------------------------------------------------

                                            Area of standard   X  Concentration of sample