Mirosław Hawrył

Separation of coumarins from Archangelica officinalis in high-performance liquid chromatography and thin-layer chromatography systems.

Complex, multicomponent mixtures are difficult to separate in a single chromatographic run. Therefore, the possibility to separate twelve coumarins from Archangelica officinalis was studied by combining a HPLC and a TLC system. HPLC optimized by the use of DryLab for Windows software was performed on RP-18 column and TLC was performed on silica plates. Fractions from the RP column were evaporated, applied on silica plate and developed in non-aqueous solvent. Possibilities of complete separation of investigated coumarins were discussed in RP and NP systems. The result of their complete separation was presented by HPLC chromatograms, DryLab simulated chromatograms and a video scan of TLC plate.

Application of normal- and reversed-phase 2D TLC on a cyanopropyl-bonded polar stationary phase for separation of phenolic compounds from the flowers of Sambucus nigra L.

Two-dimensional thin-layer chromatography on a cyanopropylbonded 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.

Retention behavior of some flavonoids in 2D-TLC systems on cyano bonded polar phases

SummaryTwo-dimensional thin layer chromatography of 16 flavonoids and 3 phenolic acids was performed in normal and reversed phase systems on plates with chemically bonded-cyanopropyl stationary phase. TheRM values in some non-aqueous and aqueous systems were utilized to optimize separation.RM values were correlated for all investigated chromatographic systems and then optimum pairs of systems were selected for use in two-dimensional TLC separations.

Two-dimensional planar chromatography of tropane alkaloids from Datura innoxia Mill.

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. Normalphase separation on silica layers was optimized for quantitative analysis of the alkaloids scopolamine and tropine and the sum of atropine and homatropine.

Analysis of Plant Material

This chapter covers the subject of instrumental thin-layer chromatographic (TLC) analysis of plant extracts. Single development techniques for the separation of plant extract fractions of various structures are reviewed. The following metabolite groups are described: polyphenolic compounds (phenolic acids, flavonoids, coumarins, lignans), terpenoids (mono-, di-, sesqui- terpenes, saponins, carotenoids), iridoids, alkaloids, and quinones. Special techniques for chromatogram development, such as: unidimensional multiple development, incremental multiple development, and bivariant multiple development and their application in plant analysis are described. Multidimensional separations and their realization by TLC are described, e.g., 2D-TLC on one plate, on bilayers, and on connected plates (graft TLC) with examples of their use in phytochemical analysis. Strategies for the separation of compounds of a wide polarity range, such as stepwise gradient development, binary development, and multiple gradient development are addressed. Also the strategies for separation of structural analogues are mentioned. The use of all the aforementioned techniques in fingerprint analysis of plant material is discussed. The advantages of using biological detection for screening of plant samples for the presence of pharmacologically active metabolites are characterized. Finally the use of image processing of TLC videoscans or photographs is mentioned.

Two-dimensional thin-layer chromatographic determination of phenolic antioxidants from Eupatorium cannabinum extracts on cyano-bonded polar stationary phases

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 RF 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.

Two-dimensional Thin Layer Chromatographic Separation of Phenolic Compounds from Eupatorium cannabinum Extracts and their Antioxidant Activity

Two dimensional thin layer chromatography on DIOL polar bonded stationary phase was performed to optimize the separation of some anioxidant phenolic compounds from Eupatorium cannabinum extracts. Ethyl-methyl ketone 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 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. Satisfactory separations of antioxidant phenolic compounds in Eupatorium cannabinum extracts were obtained by use of optimized 2D-HPTLC systems.

Separation of phenolic compounds by NP and RP two-dimensional thin layer chromatography on connected plates

Separation selectivity has been optimized in two-dimensional thinlayer 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 statis-tical data for correlation of RM values for pairs of chromatographic systems - NP diol-RP C18 or silica-RP C18. 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.

Application of densitometry to the determination of catechin in rose-hip extracts

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 F254 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. penduli-na 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.

Micro-two-dimensional TLC in search of selected Mentha sp. Extracts for their composition and antioxidative activity

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.