APPLICATION NOTES

HPTLC Identification of Black Cohosh (Cimicifuga racemosa)

F-13A

Triterpene glycosides

Sample:
In a conical flask 0.5 g powdered drug are sonicated with 5
mL methanol for 10 min, then filtered through filter paper. The
filter is washed 4 times with 1 mL methanol each. In a
volumetric flask the combined filtrates are adjusted to 10 mL
with methanol. 1 mL of the solution is transferred into a small
sample vial. This is the test solution.
Analyzing the extract and the Herbal Medicinal Product, the
quantity corresponding to 0.5 g of the powdered drug is used.
Standards (optional):
2.5 mg actein is dissolved in 10 mL ethanol water 6:4.
2 mg deoxyactein is dissolved in 10 mL methanol.
Derivatization reagent:
Anisaldehyde-sulfuric acid reagent: 10 mL of 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:

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Stationary phase:

HPTLC plates silica gel 60 F254 (Merck), 10x10 cm or

20x10 cm.

Mobile Phase:

Ethyl formiate, toluene, formic acid (30:50:20)

Sample application:

2 L test solution and standard are applied as 8 mm bands,

min. 2 mm apart, 8 mm from lower edge of plate.

Please contact CAMAG for more application notes and products for analysis of herbals!

F-13A

APPLICATION NOTES

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 (warm
air) for 5 min.

Detection:

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.

Results:
Compare to the chromatograms below:
Comparison of different samples
1

10

Comparison of extract and Herbal Medicinal Product

1

1
2
3

Serratula sp. (adulterant)

Cimicifuga racemosa
Sheng Ma (Chinese
adulterant, C. dahurica)
4 Actaea podocarpa
(Cimicifuga americana)
5 Actein
6 27-Deoxyactein
7 Cimicifuga racemosa
authenticated drug
8 Cimicifuga racemosa
authenticated drug
9 Cimicifuga racemosa
authenticated drug
10 Cimicifuga racemosa
authenticated drug

1
2
3
4
5
6
7

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Actein
27-Deoxyactein
Extract
Herbal Medicinal Product
Placebo
Actein
27-Deoxyactein

Please contact CAMAG for more application notes and products for analysis of herbals!

F-13A