Reversed-phase thin layer chromatographic determination of oleanolic and glycyrrhizinic acids in aqueous-organic and modified micellar mobile phases

Abstract

Saponins to be representatives of natural high-molecular biologically active substances are widely
used in food, pharmaceutical, cosmetic industries as natural surfactants. Analytical methods described for the
determination of saponins in commercial and natural objects are based on HPLC with mass-spectrometric
(MS), tandem MS with electrospay ionization, or UHPLC that are very expensive and considerable time
costs. The aim of this work was to investigate the possibility to separate some saponins by simple and low
cost TLC method using biodegradable and nontoxic aqua micellar mobile phases (MMP) and MMP
modified by different alcohols. Stock standard saponins solutions 2 mg/mL of glycyrrhizinic, oleanolic acids,
saponin and hederakozid (Sigma, Fluka, USA) were prepared in ethanol. The study was performed by
reversed-phase liquid chromatography in the mode of ascending TLC on commercial plates RP-18,
Polyamide-6 (both Merck, Germany), Sorbfil (Sorbopolymer, Russia), and HPLC on a column Luna C18(2)
(150 ´ 4.6 mm, 5 μm, Phenomenex, USA). The detection and quantitative measurements of chromatograms
in TLC were conducted using a Sorbfil videodensitometer (Sorbopolymer, Russia). Aqueous–organic MP
containing propanol-1, propanol-2, butanol-1, butanol-2, and also micellar mobile phases containing cationic
surfactant cetylpyridinium chloride (CPC), anionic surfactant sodium dodecyl sulfate (both from Synthesis
PAV, Russia), or nonionic surfactant Triton X-100 (Merck, Germany) were studied. The experiment showed that the best chromatographic behavior of saponins in aqua-organic mobile phases was observed for RP-18
plates, whereas in MMP phases the saponins chromatographic spots were stayed at the start line. On addition
of alcohols the spots were moved and the best separation of saponins was observed for CPC micelles with
addition of 25% of buthanol-1. The reason for such effect of alcohols is, probably, that they form hydrogen
bonds with molecules of the studied substances in the mobile phase, which reduces their retention on the
stationary phase and intensifies the mobility of saponins. The usefulness of the proposed mobile phases for
the separation and determination of oleanolic and glycyrrhizinic acids in vegetable pharmaceutical
preparations and foodstuff by TLC was shown. The results obtained by proposed TLC method are in
agreement with HPLC ones.
The retention and separation of four saponins in aqueous–alcoholic and aqueous–MMP containing
cationic, anionic and nonionic micelles of surfactants and MMP modified with alcohols was studied by
reversed-phase TLC on varying the concentration of surfactants and the nature and concentration of alcoholic
modifiers of MMP. The optimal chromatographic system based on cetylpyridinium chloride micelles and
butanol-1 was found. The proposed simple TLC procedure was used to determine oleanolic and
glycyrrhizinic acids in vegetable pharmaceutical preparations and foodstuff.