An Improved Method to Resolve Plant Saponins and Sugars by TLC

Thin-layer chromatography (TLC) plays an important role in the initial selection of mutants having a unique seed saponin composition from the germplasm collections of the subgenus Soja. In the conventional TLC procedure, the dehydrated free sugars are retained just below the major saponins and interrupt the identification of some minor saponin constituents. To resolve this problem, we developed an efficient and reliable method to move sugars from the saponin area on TLC. A developing chamber was saturated with the lower phase of chloroform:methanol:water (65:35:10, v/v) for 2?h and the TLC plates were developed in it for 50?min. Plates were then dried at 100?°C for 10?min to evaporate the excess mobile phase and developed again with 10?% H₂SO₄ for 15?min. While sulfuric acid migrates over the surface of SiO₂, sugar molecules are dehydrated and hydrophilic interactions between free sugars and SiO₂ are strongly reduced. Thus, the positions of dehydrated sugars were shifted to above the saponin area on the TLC plate. This resulted in easy recognition of the saponin composition without any discrimination. This amended protocol would be applicable to all TLC analyses in which the target components should be separate from the interrupting sugar molecules.