Łukasz Cieśla

Two-dimensional thin-layer chromatography in the analysis of secondary plant metabolites

Drugs, derived from medicinal plants, have been enjoying a renaissance in the last years. It is due to a great pharmacological potential of herbal drugs, as many natural compounds have been found to exhibit biological activity of wide spectrum. The introduction of whole plants, plant extracts, or isolated natural compounds has led to the need to create the analytical methods suitable for their analysis. The identification of isolated substances is relatively an easy task, but the analysis of plant extracts causes a lot of problems, as they are usually very complex mixtures. Chromatographic methods are one of the most popular techniques applied in the analysis of natural mixtures. Unfortunately the separation power of traditional, one-dimensional techniques, is usually inadequate for separation of more complex samples. In such a case the use of multidimensional chromatography is advised. Planar chromatography gives the possibility of performing two-dimensional separations with the use of one adsorbent with two different eluents or by using bilayer plates or graft thin-layer chromatography (TLC) technique; combinations of different multidimensional techniques are also possible. In this paper, multidimensional planar chromatographic methods, commonly applied in the analysis of natural compounds, were reviewed. A detailed information is given on the methodology of performing two-dimensional separations on one adsorbent, on bilayer plates, with the use of graft TLC and hyphenated methods. General aspects of multidimensionality in liquid chromatography are also described. Finally a reader will find a description of variable two-dimensional methods applied in the analysis of compounds, most commonly encountered in plant extracts. This paper is aimed to draw attention to the potential of two-dimensional planar chromatography in the field of phytochemistry. It may be useful for those who are interested in achieving successful separations of multicomponent mixtures by means of two-dimensional TLC.

Approach to develop a standardized TLC-DPPH test for assessing free radical scavenging properties of selected phenolic compounds

TLC-DPPH(•) test belongs to a group of frequently performed assays aimed at detection of compounds with desired activity (effect directed analysis). Despite its popularity a standard procedure has not been elaborated so far causing difficulties in comparing results obtained in different laboratories. Thus the aim of the presented research was an approach to develop a standardized procedure for assessing free radical scavenging properties of plant polyphenols. It was observed that specifically positive adsorbent (silica gel) strengthened the observed result of radical-antioxidant reaction, while polar bonded stationary phase CN-silica weakened it. Based on the observed results it was concluded that the TLC-DPPH(•) assay should be preferably performed on the surface of non-specific adsorbents (e.g.: RP-18) with the use of n-hexane for DPPH(•) dissolution. It is also proposed to document the results every 5 min after staining, as they change in time.

Investigation of antiradical activity of plant material by thin-layer chromatography with image processing

A novel, easy, and cheap technique for preliminary quantitative evaluation of antiradical activity, based on HPTLC, has been proposed. This method combines chromatographic separation of polar compounds, present in plant extracts, with data analysis by means of image processing software. Bleaching of the purple DPPH colour, caused by substances with antiradical activity, was observed and recorded using a photo camera. ImageJ, a free and open source image processing program was used for quantitative measurements. For evaluation of assay efficiency, the antiradical activity of rose flower extracts (from Rosa rugosa Thunb.) was expressed as Standard Activity Coefficients (SACs), which are relative measures of the activity to the four well known antioxidants; i.e., quercetin, gallic acid, protocatechuic acid, and Trolox. The method uses small amounts of free radical and is easily applicable - only a digital camera with freely available open source software is required.

Two-dimensional thin-layer chromatography with adsorbent gradient as a method of chromatographic fingerprinting of furanocoumarins for distinguishing selected varieties and forms of Heracleum spp.

There are a lot of taxonomic classifications of the genus Heracleum, and many authors indicate they need revision. Morphological identification is difficult to perform, as there are only few characteristic differences between each Heracleum species, varieties and forms. Furanocoumarins are characteristic compounds for the Apiaceae family, and they can be found in the whole genus in large quantities. Despite this fact, it is difficult to use the furanocoumarin profiles of plants, for their discrimination, as furanocoumarins are difficult to separate, due to their similar chemical structures and physicochemical properties. In this paper, a new, simple method is proposed for the discrimination of selected species, varieties and forms of the genus Heracleum. Thin-layer chromatography (TLC) with an adsorbent gradient (unmodified silica gel+octadecylsilica wettable with water) enables complete separation of the structural analogues. The proposed method gives the possibility to distinguish selected species, varieties and forms of the Heracleum genus, as they produce distinctive furanocoumarin fingerprints. The method is characterised by high specificity, precision, reproducibility and stability values. It is for the first time that graft TLC is used for constructing fingerprints of herbs. The complete separation of ten structural analogues, by combining gradient TLC with the unidimensional multiple development technique, has not been reported yet.

The start-to-end chemometric image processing of 2D thin-layer videoscans

The purpose of the research was to recommend a unified procedure of image preprocessing of 2D thin layer videoscans for further supervised or unsupervised chemometric analysis. All work was done with open source software. The videoscans saved as JPG files underwent the following procedures: denoising using a median filter, baseline removal with the rollerball algorithm and nonlinear warping using spline functions. The application of the proposed procedure enabled filtration of random difference between images (background intensity changes and spatial differences of the spots location). After the preprocessing only spot intensities have an influence on the performed PCA or other techniques. The proposed technique was successfully applied to recognize the differences between three Carex species from the 2D videoscans of the extracts. The proposed solution may be of value for the any chemometric task--both unsupervised and supervised.

Development of chromatographic and free radical scavenging activity fingerprints by thin-layer chromatography for selected Salvia species.

INTRODUCTION Plant-derived free radical scavengers have become the subject of intensive scientific interest. Recently, the concept of coupling chromatographic fingerprints with biological fingerprinting analysis has gained much attention for the quality control of plant extracts. However, identification of free radical scavenging activity of each single compound in a complex mixture is a difficult task. Thin-layer chromatography with post-chromatographic derivatisation with the methanol solution of DPPH can be a valuable tool in such analyses. OBJECTIVE Development of chromatographic and free radical scavenging fingerprints of nineteen Salvia species grown and cultivated in Poland. METHODOLOGY Chromatography was performed on the silica gel layers with use of two eluents, one for the resolution of the less polar compounds, and the other one for the resolution of the medium and highly polar ones. The plates were sprayed with the vanillin-sulfuric acid reagent to produce chemical fingerprints, and with DPPH solution to generate free radical scavenging fingerprints. RESULTS With four Salvia species, it was revealed that their strong free radical scavenging properties are not only due to the presence of polar flavonoids and phenolic acids, but also due to the presence of several free radical scavengers in the less polar fraction. Because of the similarities in both the chromatographic and the free radical scavenging fingerprints, S. triloba can be introduced as a possible equivalent of the pharmacopoeial species, S. officinalis. CONCLUSION Fingerprints developed in the experiments proved useful for the analysis of complex extracts of the different Salvia species.

Two-Dimensional thin-layer chromatography of structural analogs. Part I: Graft TLC of selected coumarins

Coumarins are natural, biologically active substances, normally found in complex mixtures. Unfortunately their separation causes many difficulties, because of to their similar chemical structure and physicochemical properties. A new, reliable method has been established for analysis of coumarin fractions present in selected fruit extracts. The substances were separated in chromatographic systems that enabled use of orthogonal separation mechanisms (i.e. characterized by different selectivity). The greatest selectivity differences were obtained by use of two chromatographic systems — first dimension CN-silica with 30% ACN in H2O as mobile phase (triple developed) and second dimension SiO2 with 35% AcOEt in n -heptane as mobile phase (triple developed), or first dimension SiO2 with 35% AcOEt in n -heptane as mobile phase (triple developed) and second dimension RP-18 with 55% MeOH in H2O as mobile phase. The aforementioned two-dimensional TLC systems were used for separation of coumarin fractions present in extracts from Archangelica officinalis, Pastinaca sativa and Heracleum sphondylium fruits.

Application of Hydrophilic Interaction TLC Systems for Separation of Highly Polar Glycosidic Compounds from the Flowers of Selected Verbascum Species

Separation of highly polar compounds, for example, iridoids and triterpene saponins, present in natural samples is a challenging task. Because of their nonvolatility and the lack of chromophores, their analysis by means of gas chromatography or high-performance liquid chromatography (HPLC) is difficult. The use of normal as well as reversed-phase systems fails to resolve highly polar compounds that are strongly adsorbed on polar stationary phases and poorly retained on alkyl-bonded stationary phases usually used in HPLC. Because of these facts, separation has been performed in hydrophilic interaction systems by means of thin-layer chromatography (TLC). Planar chromatography offers also a possibility to use different derivatizing agents to visualize the resolved compounds. Another problem encountered in the analysis of such compounds present in plant extracts is that they occur as multicomponent mixtures. In this article, two-dimensional (2D) TLC systems were used for the resolution of compounds present in the polar fractions of the different Verbascum spp. flower extracts. TLC separations were performed on silica gel plates, with two different mobile phases used in the perpendicular directions. First, the analyzed samples were developed with AcOEt-MeOH-H2 O-25% aqueous NH3 (55:35:9:1, v/v/v/v) and redeveloped with MeOH-AcOEt-H2 O-HCOOH (10:90:26:22, v/v/v/v) in a perpendicular direction. The resolved compounds were visualized using the vanillin-sulfuric acid reagent. The obtained videoscans were further used for preliminary comparative studies of the investigated species. It is for the first time that 2D hydrophilic interaction systems, characterized with different pH values, are being applied for the analysis of highly polar compounds present in selected Verbascum spp. flower extracts. Anew approach of using an image-processing program for the comparative studies and for method validation is also presented.

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.

COMPARISON OF TWO TLC-DPPH•-IMAGE PROCESSING PROCEDURES FOR STUDYING FREE RADICAL SCAVENGING ACTIVITY OF COMPOUNDS FROM SELECTED VARIETIES OF MEDICAGO SATIVA

TLC-DPPH• test coupled with different image processing procedures have become an interesting technique to screen complex samples for the presence of free radical scavengers. Several procedures have already been published showing the possibility of obtaining quantitative data from TLC plates derivatized with methanolic solution of relatively stable free radical DPPH• (2,2-diphenyl-1-picrylhydrazyl). In this study two different image processing programs, ImageJ and Sorbfil TLC Videodensitometer, have been applied to study direct antioxidant properties of compounds separated from extracts obtained from selected varieties of Medicago sativa. M. sativa var. Radius possessed the greatest amount of compounds with direct antioxidant properties. The activity of the individual compounds has been compared to the activity of rutin, a flavonoid with well recognized antioxidant properties. Neither of the separated compounds was as active as the standard.