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  • Author or Editor: Mirosław Hawrył x
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Thin-layer chromatography and densitometry have been used for qualitative and quantitative analysis of catechin in methanolic extracts from 15 taxa of Rosa L. hips. Chromatography was performed on glass silica gel 60 F 254 plates with ethyl acetate-water-formic acid-acetic acid, 25 + 4 + 0.6 + 0.4 ( v/v , upper phase), as mobile phase. Chromatograms were scanned in UV light at λ = 254 nm. The amount of complex epimers of catechin was determined as (+)-catechin equivalent in the rose extracts investigated. The concentrations of (+)-catechin ranged from 135 to 930 μg g −1 . The highest catechin content was found in R. gallica L., R. pendulina L., R. tomentosa Sm., R. villosa L. subsp. mollis R. Keller et Gams., and R. dumetorum Thuill. The densitometric method was found to be simple, rapid, and convenient for routine quantitative analysis of catechin in rose hips and may be applied to determination of the compound in other plant materials.

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Two-dimensional thin-layer chromatography on a cyanopropyl-bonded polar stationary phases has been used to separate phenolic compounds occurring in Flos Sambuci L. The separation was realized by development of the chromatographic plate in two directions with different mobile phases in each direction. Optimum systems were selected by analysis of retention data obtained for a variety of concentrations of polar solvents in a non-polar diluent ( n -hexane) for NP TLC and polar solvents in water for RP TLC, both on thin layers of cyanopropyl-bonded polar adsorbents. The first development was performed by with a non-aqueous mobile phase and the second with an aqueous mobile phase. Eight flavonoids and three phenolic acids were separated by use of the method described.

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Separation selectivity has been optimized in two-dimensional thin-layer chromatography (2D TLC) by connecting diol or silica plates (on which NP chromatograms were developed) to RP-18W plates (on which reversed-phase chromatograms were developed). Retention of test substances was investigated to select optimum chromatographic systems for separation of selected phenolic compounds by 2D TLC. Selection of optimal mobile phases was performed on the basis of plots of retention against mobile phase composition. The next step of the optimization was calculation of statistical data for correlation of R M values for pairs of chromatographic systems — NP diol-RP C 18 or silica-RP C 18 . Orthogonal chromatographic systems were selected on the basis of these correlations and used to separate phenolic compounds present in plant extracts. For example, extracts from Polygonum sp. and Verbascum sp. were separated by use of optimum 2D TLC systems.

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Two-dimensional thin layer chromatography on cyano-bonded polar stationary phase was performed to optimize the separation of some antioxidant phenolic compounds from Eupatorium cannabinum extracts. Propan-2-ol mixed with n-heptane and ethyl acetate mixed with n-heptane were used as non-aqueous mobile phases in normal phase separations (1st direction of development in 2-dimensional high-performance thin-layer chromatography (2D-HPTLC mode)) and methanol mixed with water was used as a mobile phase in reversed phase (2nd direction of development in 2D-HPTLC mode). The plates were sprayed by use of Merck TLC sprayer using 2-(diphenylboryoxy)-ethylamine and PEG4000 (Merck, Darmstadt, Germany) or DPPH and photographed in CAMAG Cabinet UV lamp at 254 nm and 365 nm by use of Fuji 8 mpx camera. The plots RF non-aqueous mobile phase vs R F aqueous mobile phase were prepared to find optimal orthogonal 2D-TLC systems for the separation of investigated test compounds and then extracts from Eupatorium cannabinum. Satisfactory separations of antioxidant phenolic compounds in E. cannabinum extracts were obtained by use of optimized 2D-HPTLC systems.

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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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Seven different Scutellaria species were analyzed using the extraction procedure (Soxhlet apparatus, dichloromethane, and methanol as solvents) and thin-layer chromatography method. Selected standards of flavonoids and phenolic acids (caffeic acid, chlorogenic acid, ferulic acid, baicalein, wogonin, baicalin, chrysin, quercetin, scutellarin, hesperetin, hesperidin, apigenin, luteolin, rutin, and kaempferol) were separated using silica gel thin-layer chromatography (TLC) plates with the mobile phase consisting of ethyl acetate—toluene—formic acid (5:4.9:0.1, v/v) for dichloromethane and methanolic extracts. Dichloromethane extracts were also developed using cyanopropyl-bonded silica gel with the following mobile phases: propan-2-ol—n-heptane—formic acid (5:4.9:0.1, v/v) and methanol—water—formic acid (6:3.9:0.1, v/v), and after drying, they were sprayed using the anisaldehyde reagent. In the case of methanolic extracts, the same non-aqueous eluent was used and the aqueous eluent consisting of methanol—water—formic acid (4:5.9:0.1, v/v). The presence of selected standards in Scutellaria species was confirmed. The similarities between the obtained fingerprint chromatograms were performed using chemometric methods, the similarity coefficients (Pearson’s correlation coefficient, determination coefficient, and congruence coefficient), distance indices (Euclidean distance, Manhattan distance, and Chebyshev’s distance), and multi-scale structural similarity (MS-SSIM).

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Ten methanolic extracts of selected Cirsium species were analyzed using two-dimensional thin-layer chromatography (2D-TLC) system with octadecyl reversed-phase (RP-18) chromatographic plate as the stationary phase and two eluents: nonaqueous, consisting of 2-butanone‒toluene‒acetic acid (4.5:5:0.5, v/v) used in the first direction of developing, and aqueous, consisting of methanol—water—formic acid (4:5:1, v/v) used in the second direction. The Naturstoff reagent was used for the derivatization of some phenolic compounds. Five selected standards were analyzed under the same chromatographic conditions, and their retention factor values were used for the confirmation of their presence on selected Cirsium chromatograms. Photographs of ten chromatograms were treated using the ImageJ program. 2D-TLC analysis was also performed to obtain the fingerprint chromatographic profiles of the studied methanolic extracts. The experimental data were objected to principal component analysis (PCA), and the PC2 vs. PC3 graphs were created. Based on the PCA results, the similarity between the selected Cirsium species was confirmed.

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Fourteen different Achillea species were extracted using the Soxhlet apparatus with dichloromethane and then methanol as solvents. The obtained dichloromethane and methanolic extracts were analyzed using thin-layer chromatography (TLC) and high-performance thin-layer chromatography (HPTLC) to obtain fingerprint profiles. The first chromatographic system consisted of silica gel as adsorbent, a mixture of toluene—ethyl acetate—formic acid (5:4.9:0.1, v/v) as the mobile phase and anisaldehyde as the derivatization reagent. The second adsorbent was cyano (CN) silica gel with non-aqueous (2-propanol and n-heptane [5:5, v/v], both for dichloromethane and methanolic extracts) and aqueous (methanol and water [6:4, v/v] for dichloromethane extracts and methanol and water [4:6, v/v] for methanolic extracts) mobile phases. Anisaldehyde (for dichloromethane extracts) and Naturstoff (for methanolic extracts) derivatization reagents were used. The obtained chromatograms were processed to obtain selected images using the ImageJ program. The similarity between the studied Achillea species was confirmed using the chemometric methods (principal component analysis [PCA], similarity, and distance measures).

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Twenty land mosses were extracted by double maceration and ultrasonic extraction techniques using the mixture of 80% ethanol and water. The obtained extracts were analyzed using thin-layer chromatography (TLC) with silica gel (the mobile phase was consisted of ethyl acetate–formic acid–acetic acid–water, 14.0:1.5:1.5:2.0, v/v) and RP-18 (the composition of eluent methanol–water–formic acid, 7.0:2.5:0.5, v/v) chromatographic plates. After developing and drying, the plates were sprayed using the Naturstoff reagent, and after drying, the chromatograms were photographed and the obtained images were processed using the ImageJ program. Principal component analysis was performed to confirm the chemical similarity between the studied moss extracts. The cytotoxic activity of the ethanolic extracts of Bryophyta species was studied using the cell lines CCRF/CEM and CEM. For most of the moss samples, the cytotoxic activity was confirmed.

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JPC - Journal of Planar Chromatography - Modern TLC
Authors: Maria Gadzikowska, Anna Petruczynik, Monika Waksmundzka-Hajnos, Mirosław Hawrył and Grzegorz Jóźwiak

Plant extracts from Datura innoxia Mill. contain tropane alkaloids which are pharmacologically active, and compounds used in medicine are isolated industrially from the plant. Control of the composition and content of these alkaloids is, therefore, an important task. Thin-layer chromatography is an effective method for analysis of these compounds, because they are difficult to detection with UV light. In the work discussed in this paper separation of closely related alkaloids was achieved by two-dimensional TLC in optimized NP and RP systems by use of multiphase plates. The work enabled identification of tropane alkaloids present in plant extracts. Normal-phase separation on silica layers was optimized for quantitative analysis of the alkaloids scopolamine and tropine and the sum of atropine and homatropine.

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