Separation of Steviol Glycosides by HPTLC: Introducing a New Stevia Extract Reference Material

Debora Frommenwiler, Application Scientist (CAMAG); Melanie Broszat, Scientific Business Development Manager (CAMAG);
Monika Bäumle, Product Manager TLC; Matthias Nold, Product Manager Reference Materials

High-Performance Thin-Layer ChromatographyHPTLC (High-Performance Thin-Layer Chromatography) is a fast and efficient tool to create molecular fingerprints of complex chemical mixtures. Therefore it is particularly well suited for the analysis of plants and plant derived products. In a series of articles in Analytix and Analytix Reporter journals,1-4 we showed several examples of HPTLC of plants used as dietary supplements or as herbal medicinal products such as Ginkgo, Ginseng or St. John’s Wort.5 That series is continued here with a fingerprint method for Stevia rebaudiana leaves, including the analysis of our new stevia extract reference material.

Extracts from the leaves of the Stevia rebaudiana plant have a long tradition of being used as a sweetener. Native tribes of Latin America have known and consumed it for centuries because of the sweet taste. Stevia extracts have approximately 300 times more intense sweetness than sucrose while only having a negligible effect on blood glucose. Therefore, in recent years, the plant has increasingly been used in other parts of the world as an alternative to artificial sweeteners.

Stevia is FDA approved as a dietary supplement and rebaudioside A is considered to be “Generally Recognized As Safe (GRAS)”. The European Community has allowed the use of steviol glycosides as food additives since December 2011.

The WHO defined the acceptable daily intake of steviol glycosides at 4 mg per kg body weight.6

We recently launched a new extract reference material, developed and manufactured by HWI pharma services GmbH in Rülzheim, Germany:


Description Quantified Components Qualitatively Confirmed Components Package Size Cat. No.
Stevia extract Stevioside Rebaudiosides A, B, C and D, Dulcoside A, Rubusoside, Steviolbioside, Stevioside 500 mg 06295001


This new product complements our range of plant extract reference materials designed for use as a rapid identification and quantification method for typical constituents of plants used as food additives or as herbal medicinal products (see the complete offer at

The Stevia extract reference material is provided with comprehensive documentation including a quantitative value for the major component Stevioside as well as qualitative conformation of various other constituents (Rebaudiosides A, B, C and D, Dulcoside A, Rubusoside, Steviolbioside, Stevioside). In addition to an HPLC method including a chromatogram with assigned peak identities, the documentation also contains an HPTLC method according to [5].

For the analysis, Supelco® HPTLC plates and reagents have been used. The analytical standards of the pure steviol glycosides are listed below. Please find a comprehensive listing of our entire phytochemical standards range at

HPTLC method

The scope of the method is the identification of a Stevia rebaudiana leaf dry extract reference material based on HPTLC fingerprints of steviol glycosides obtained with the HPTLC method by Wald and Morlock 20175 by comparison with the fingerprint of Stevia leaf. Additionally, chemical reference substances were used for identification of the zones of the chromatogram.


Automatic TLC Sampler (ATS 4), Automatic Developing Chamber (ADC 2), Chromatogram Immersion Device 3, TLC Plate Heater 3, TLC Visualizer, visionCATS (the software offers a Method Library that includes an SOP for each method, an instrument method, and a comparison file with reference images).


Extract: 50 mg were suspended in 50 mL of methanol and sonicated for 10 min. The suspension was centrifuged, and the supernatant used.

Leaf: 0.5 g of powdered leaf was suspended in 30 mL of water and boiled for 10 min. The solution was filtered into a 50 mL volumetric flask and the volume was made up with water.


Standard solutions were prepared in a concentration of 0.3 mg/mL in methanol. (Note: This is 9.09–fold more concentrated than in [5])


Chromatography according to USP <203>
Stationary phase HPTLC Si 60 F254, 20 x 10 cm (1.05642)
Sample application Application with ATS 4, 10 tracks, band length 8 mm, track distance 11.4 mm, distance from left edge 20 mm, distance from lower edge 8 mm, application volume 2 μL for test solutions and standards
Developing solvent Ethyl acetate, methanol and formic acid 93:40:1 (v/v/v)
Development In the ADC 2 without chamber saturation and after conditioning at 33% relative humidity for 10 min using a saturated solution of magnesium chloride
Developing distance 70 mm (from the lower edge)
Plate drying 5 min in the ADC 2
Documentation With the TLC Visualizer under UV 366 nm and white light after derivatization
Derivatization Reagent name: 2-Naphthol
Reagent preparation (dipping) 2 g of 2-naphthol in 180 mL of ethanol and 12 mL of 50% sulfuric acid
Reagent use The plate was immersed into 200 mL of 2-naphthol reagent using the Chromatogram Immersion Device (immersion time 0 s and immersion speed 3 cm/s) and then heated at 120°C for 5 minutes


The derivatized plates were viewed under UV light at 366 nm and white light (Figure 1). In the fingerprint of the HWI extract (track 8), zones corresponding in color and position to those of the standards Rebaudioside A, C, Stevioside, Rebaudioside B and Dulcoside A (which are co-eluting), and Steviolbioside are seen. The fingerprint is similar to those of S. rebaudiana leaf (tracks 9 and 10). Rebaudioside D is only seen in the fingerprint of the leaf, particularly under UV 366 nm (very faint zone).


Figure 1. HPTLC chromatograms after derivatization under UV 366 nm (top) and white light (bottom). Track 1: Rebaudioside D; 2: Rebaudioside A; 3: Rebaudioside C; 4: Stevioside; 5: Rebaudioside B; 6: Dulcoside A; 7: Steviolbioside; 8: Stevia rebaudiana leaf dry extract reference material (HWI); 9: Stevia rebaudiana leaf 1; 10: Stevia rebaudiana leaf 2

HPTLC chromatograms after derivatization under UV
HPTLC chromatograms after derivatization under UV



  1. Analytix 5 (2016): HPTLC Fingerprint Applications for Ginkgo Biloba
  2. Analytix 1 (2017): Fingerprint Applications for Hypericum perforatum
  3. Analytix Reporter 2 (2018): Fingerprinting of Medicinal Plants with TLC
  4. Analytix Reporter 3 (2018): HPTLC Application for Passiflora incarnata
  5. Wald JP, Morlock G. Quantification of steviol glycosides in food products, Stevia leaves and formulations by planar chromatography, including proof of absence for steviol and isosteviol. Journal of Chromatography A, 2017.
  6. WHO Food Additives Series 54; 2006; page 117