APPLICATION NOTES

HPTLC Identification of African Prune Bark (Pygeum africanum) F-20

Sample:
0.5 g milled sample are sonicated with 5 mL ethanol for
5 min. The mixture is filtered through a 0.2 m syringe filter.

Standard (optional):
2 mg -sitosterol are dissolved in 10 mL methanol

Derivatization reagent:
Anisaldehyde-sulfuric acid reagent: 10 mL sulfuric acid are
carefully added to an ice-cooled mixture 170 mL methanol
and 20 mL acetic acid. To this solution 1 mL anisaldehyde is
added.
Note: This reagent is colorless and should be stored in the
refrigerator. If a color develops the reagent must be
discarded.

Chromatographic conditions:
Stationary phase: HPTLC plates silica gel 60 F254 (Merck), 10x10 cm or
20x10 cm.
Mobile Phase: Toluene, ethyl acetate (8:2)
Sample application: 3 L each of test solution and standard are applied as 8 mm
bands, min 2 mm apart, 8 mm from lower edge of plate.
Development: 10x10 cm or 20x10 cm Twin Trough Chamber, saturated for
20 min (filter paper), 5 mL (respectively 10 mL) developing
solvent per trough, developing distance 70 mm from lower
edge of plate. The plate is then dried with a hair dryer (cold
air) for 5 min.
Detection: a) UV 366 nm

09/03 Please contact CAMAG for more application notes and products for analysis of herbals! F-20
APPLICATION NOTES

b) Anisaldehyde-sulfuric acid reagent: the plate is immersed

in the reagent for 1 s, then heated at 100C for 2-5 min.
Evaluation in white light.
c) Examine the derivatized plate under UV 366 nm

Results:
Compare to the chromatograms below:
Image prior to derivatization
1 2 3 4 5 6 7 8 9 10 11 12

a) UV 366

Images after derivatization

1 2 3 4 5 6 7 8 9 10 11 12
HPTLC of African Prune:

1-5, 7-11: Prunus africanus bark

12: Liquid sample
6: -sitosterol

E: Ethanol extraction
D: Dichloromethane
extraction

b) White light

1 2 3 4 5 6 7 8 9 10 11 12

c) UV 366

09/03 Please contact CAMAG for more application notes and products for analysis of herbals! F-20