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APPLICATION NOTES

HPTLC Identification of Echinacea (E. purpurea, E. angustifolia, E. pallida) F-24A

Phenolics

Sample:
1 g milled sample is sonicated with 10 mL methanol for 5 min.
The extract is filtered through a 0.2 m syringe filter.
Standards (optional):
2 mg echinacoside, 2 mg cynarine, 1 mg chlorogenic acid,
1 mg caffeic acid, 0.5 mg caftaric acid, and 0.5 mg chicoric
acid are dissolved in 10 mL methanol each.

Derivatization reagent:
Natural Products reagent (NP reagent): 1 g diphenylborinic
acid aminoethylester is dissolved in 200 mL ethyl acetate.

Chromatographic conditions:
Stationary phase: HPTLC plates silica gel 60 F254 (Merck), 10x10 cm or
20x10 cm.
Mobile Phase: Ethyl acetate, ethylmethyl ketone, formic acid, water
(15:9:3:3)
Sample application: 5 L of test solution and 2 L of each standard are applied as
8 mm bands, at least 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 60 mm from lower
edge of plate. The plate is then dried with a hair dryer (cold
air) for 5 min.
Detection: NP reagent: the plate is heated at 100C for 3 min, then
dipped while still hot in NP reagent and dried in a stream of
cold air. Examination under UV 366 nm.

11/03 Please contact CAMAG for more application notes and products for analysis of herbals! F-24A
APPLICATION NOTES

Results:
Compare to the chromatograms below:

Images after derivatization, UV 366 nm

Roots
1 2 3 4 5 6 7 8 9 1: Echinacea purpurea root
2: Echinacea pallida root
3: Echinacea angustifolia root
4: Echinacea atrorubens root
5: Caftaric acid, cynarine, chicoric
acid (with increasing Rf)
6: Echinacoside, chlorogenic acid,
caffeic acid (with increasing Rf)
7: Liatris punctata root (adulterant)
8, 9: Parthenium integrifolium root
Echinacea purpurea root contains caftaric and chicoric acids. (adulterant)
Echinacea pallida root contains echinacoside.
Echinacea angustifolia root contains echinacoside and
a low amount of cynarine.
Echinacea atrorubens root shows none of the markers.
The profiles of the adulterants dont match any of the standards.

Herb and achenes


1 2 3 4 5 6 7 1: Echinacea purpurea herb
2: Echinacea angustifolia herb
3: Caftaric acid, cynarine, chicoric acid (with
increasing Rf)
4: Echinacoside, chlorogenic acid, caffeic
acid (with increasing Rf)
5: Echinacea purpurea achenes
6: Echinacea pallida achenes
7: Echinacea angustifolia achenes

Echinacea purpurea herb contains caftaric and chicoric acid.


Echinacea pallida herb shows low amount of caftaric acid (the band at high Rf is slightly lower than
that of chicoric acid).
Echinacea purpurea achenes contain no distinct zone.
Echinacea pallida achenes show a low amount of chlorogenic acid (the band at high Rf is slightly
lower than that of chicoric acid).
Echinacea angustifolia achenes contain chlorogenic acid.

11/03 Please contact CAMAG for more application notes and products for analysis of herbals! F-24A