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Two-dimensional separations were performed on polar bonded stationary phase of type cyanopropyl-silica and diol-silica by use of non-aqueous eluents (polar modifier dissolved in n-heptane) as the first direction eluents and aqueous eluents (organic modifier — MeOH dissolved in water) as the second direction eluents. The chromatographic process was performed in micro scale using 5 × 5 cm plates, small volumes of eluents, and 10 μL of plant extracts to obtain satisfying separation. Plates developed in horizontal chambers were dried and observed in UV light (254 nm and 366 nm) photographed by digital camera and derivatized by 1,1-diphenyl-2-picrylhydrazyl (DPPH) to detect antioxidants (free radical scavengers) or derivatized by Naturstoff reagent to detect phenolic compounds (characteristic luminescence of some phenolic compounds). The above experiments give the possibility to construct fingerprints for investigated Mentha species and varieties and preparations containing the herb. It can be used in quality control of the plant material and its antioxidative activity.

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Micro-thin-layer chromatography in two dimensional (2D-mTLC) mode in normal and reversed phase systems by use of diol bonded stationary phase was applied to make fingerprints of 11 species of Mentha genus and two finished pharmaceutical products.

Nonaqueous eluents (propan-2-ol or ethyl acetate dissolved in n-heptane) were used in normal phase systems. Mixtures of acetonitrile with water were used in reversed phase chromatographic systems.

Optimization of one dimensional systems was performed by determining of R F vs. composition of mobile phases dependencies for standards occurring in various species of Mentha. Most selective eluents were chosen to optimize two-dimensional systems by creating R F in normal-phase (NP) systems vs. R F in reversed-phase (RP) systems correlations.

2D-mTLC on diol polar bonded stationary phase were optimized to separate phenolic compounds and make fingerprints of examined plant materials and this method was never applied earlier in the chromatographic analysis.

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