HomeSmall Molecule HPLCSeparation of Steviol Glycosides by HPTLC: Introducing a New Stevia Extract Reference Material

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

Debora Frommenwiler, Melanie Broszat, Monika Bäumle, Matthias Nold

Cup of coffee with Stevia leaf

HPTLC (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:

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])


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).

HPTLC chromatograms after derivatization

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



2016. HPTLC Fingerprint Applications for Ginkgo Biloba. Analytix 5.
2017. Fingerprint Applications for Hypericum perforatum. Analytix 1.
2018. Fingerprinting of Medicinal Plants with TLC. Analytix Reporter 2.
2018. HPTLC Application for Passiflora incarnata. Analytix Reporter 3.
Wald JP, Morlock GE. 2017. 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. 1506:109-119.
2006. WHO Food Additives Series 54.117.