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
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
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