8/14/2019 F-24a_echinacea_phenolics HPTLC Identification of Echinacea.pdf

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

APPLICATION NOTES

HPTLC Identification of Echinacea(E. purpurea, E. angustifolia, E. pallida)

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 chicoricacid 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 or20x10 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 as8 mm bands, at least 2 mm apart, 8 mm from lower edge ofplate.

Development: 10x10 cm or 20x10 cm Twin Trough Chamber, saturated for

20 min (filter paper), 5 mL (respectively 10 mL) developingsolvent per trough, developing distance 60 mm from loweredge of plate. The plate is then dried with a hair dryer (coldair) for 5 min.

Detection: NP reagent: the plate is heated at 100C for 3 min, thendipped while still hot in NP reagent and dried in a stream ofcold air. Examination under UV 366 nm.

F-24A

Phenolics

8/14/2019 F-24a_echinacea_phenolics HPTLC Identification of Echinacea.pdf

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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

Echinacea purpurearoot contains caftaric and chicoric acids.

Echinacea pallidaroot contains echinacoside.

Echinacea angustifolia rootcontains echinacoside anda 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

Echinacea purpureaherb contains caftaric and chicoric acid.

Echinacea pallidaherb shows low amount of caftaric acid (the band at high Rf is slightly lower thanthat of chicoric acid).

Echinacea purpureaachenes contain no distinct zone.

Echinacea pallidaachenes show a low amount of chlorogenic acid (the band at high Rf is slightlylower than that of chicoric acid).

Echinacea angustifolia achenescontainchlorogenic acid.

1: Echinacea purpurearoot

2: Echinacea pallidaroot

3: Echinacea angustifoliaroot

4: Echinacea atrorubensroot

5: Caftaric acid, cynarine, chicoricacid (with increasing Rf)

6: Echinacoside, chlorogenic acid,caffeic acid (with increasing Rf)

7: Liatris punctataroot (adulterant)

8, 9: Parthenium integrifoliumrootadulterant

1 2 3 4 5 6 7 8 9

1: Echinacea purpureaherb

2: Echinacea angustifoliaherb

3: Caftaric acid, cynarine, chicoric acid (withincreasing Rf)

4: Echinacoside, chlorogenic acid, caffeicacid (with increasing Rf)

5: Echinacea purpureaachenes

6: Echinacea pallidaachenes7: Echinacea angustifoliaachenes

1 2 3 4 5 6 7