APPLICATION NOTE...HPTLC profiles under UV 254 nm (A), UV 366 nm (B), and white light (C) prior to derivatization, and under UV 366 nm after derivatization (D), and white light after

APPLICATION NOTE

NOTE: The presented results are to be regarded as examples only!Please contact CAMAG for more application notes and products!

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A-127.1

Identification of Rhodiola crenulata roots and rhizomes (Rhodiola crenulata (Hook. f. & Thomson) H. Ohba)

Keywords

Rhodiola root, Dietary supplement, USP, HPTLC

Introduction

Rhodiola crenulata root and rhizome consists of the dried roots and rhizomes of Rhodiola crenulata (Hook. f. & Thomson) H. Ohba (alt. name Sedum crenulatum Hook. f. & Thomson) (Fam. Crassulaceae), collected after the scape withers in autumn. The drug is nowadays a popular dietary supplement but may also be considered an adulterant of Rhodiola rosea, a drug used in traditional medicines.

Scope

This method identifies dried root and rhizome collected after the scape withers in autumn of Rhodiola crenulata (Hook. f. & Thomson). H. Ohba by HPTLC fingerprint, and discriminates root and rhizome of Rhodiola rosea L. as well as Sedum roseum (L.) Scop. and Sedum quadrifidum Pall. The method is included in the USP Herbal Medicine Compendium.

Recommended devices

Automatic TLC Sampler (ATS 4), Automatic Developing Chamber (ADC 2), Derivatizer, TLC Visualizer, visionCATS software

Samples

100.0 mg/mL of finely powdered sample in methanol, sonicate for 10 min, centrifuge, and use the clear supernatant

Standards

Standard solution A: 2 mg/mL of USP Salidroside RS and 1 mg/mL of USP Rosavin RS in methanol.Standard solution B: 50 mg/mL of USP Rhodiola crenulata Root and Rhizome Dry Extract RS in methanol.Optional: 1 mg/mL of rosin in methanol

APPLICATION NOTE



Chromatography

Stationary Phase HPTLC Si 60 F254, 20 x 10 cm (Merck)

Sample application 5 µL of each standard and sample solution are applied as bands with ATS 4, 15 tracks, band length 8 mm, track distance 11.4 mm, distance from left edge 20 mm, distance from lower edge 8 mm

Developing solvent Ethyl acetate, methanol, water, formic acid 77:13:10:2 (v/v/v/v)

Development In the ADC 2 with saturated chamber (20 min, with a filter paper). Plates are activated at 33% relative humidity for 10 min using a saturated solution of magnesium chloride (MgCl2).

Temperature 22 ± 5°C

Developing distance

70 mm (from the lower edge)

Plate drying Drying 5 min in the ADC 2

Documentation With the TLC Visualizer:A) Underivatized, under UV light at 254 nmB) Underivatized, under UV light at 366 nm C) Underivatized, under white lightD) Derivatized, under UV light at 366 nmF) Derivatized, under white light

Derivatization Name: Aniline-diphenylamine-phosphoric acid reagentPreparation: Dissolve 1 g of diphenylamine in 40 mL of acetone, add 1 mL of aniline, and mix. Carefully add 7.5 mL of phosphoric acid, and mix.Use: Spray (Derivatizer: 3 mL, yellow nozzle, level 6), heat at 120°C for 5 min

APPLICATION NOTE



Results

System Suitability Test (SST) under UV 366 nm after derivatization• Rosavin: orange zone at RF ~ 0.22• Salidroside: dark blue zone at RF ~ 0.40

Identification of Rhodila crenulata roots and rhizomes

HPTLC profiles under UV 254 nm (A), UV 366 nm (B), and white light (C) prior to derivatization, and under UV 366 nm after derivatization (D), and white light after derivatization (E).

Track Sample Origin1 USP Rosavin RS (1.0 mg/mL), USP Salidroside RS (2.0 mg/mL) (with

increasing RF)--

2 Rosin (1.0 mg/mL) --3 USP Rhodiola crenulata Root and Rhizome Dry Extract RS (50 mg/mL) --

4–8 Powdered Rhodiola crenulata root and rhizome (100 mg/mL) Unknown9 Powdered Rhodiola rosea root and rhizome (100 mg/mL) Unknown

10 Powdered Rhodiola rosea root and rhizome, questionable (100 mg/mL) Unknown11 Powdered Sedum roseum (L.) Scop. [syn. Rhodiola sachalinensis Boriss.]

(100 mg/mL)Unknown

12 Powdered Sedum quadrifidum Pall. [syn. Rhodiola quadrifida (Pall.) Fisch. & C.A.Mey.] (100 mg/mL)

Unknown

APPLICATION NOTE



IdentificationCompare result with reference images. The fingerprint of the test solution prepared from the sample is similar to those obtained from the corresponding botanical reference materials. Additional faint zones may be present.

The Standard solution shows two zones in the lower half of the chromatogram. The zone with a higher RF is due to salidroside, and the zone with a lower RF is due to rosavin.

The Sample solution exhibits a zone corresponding in RF and color to salidroside in the Standard solution. In the Sample solution, no zone corresponding in RF and color to rosavin is observed (a distinction from Rhodiola rosea). The Sample solution exhibits additional zones, including two zones below salidroside but above the position corresponding to rosavin, some yellow zones in the upper half of the chromatogram, and a couple of zones close to the starting position.

Documents

APPLICATION NOTE...HPTLC profiles under UV 254 nm (A), UV 366 nm (B), and white light (C) prior to derivatization, and under UV 366 nm after derivatization (D), and white light after