Authors:Anna Hawrył, Ryszard Świeboda, Mirosław Hawrył, and Agata Ziobro
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.
values for pairs of chromatographic systems has been used for practical separation of a mixture of eight cephalosporins by two-dimensional thin-layer chromatography on silica gel layers. Plates were scanned and videoscanned to show the real picture of separation.
Authors:Mirosław Hawrył and Monika Waksmundzka-Hajnos
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
values for pairs of chromatographic systems — NP diol-RP C
or silica-RP C
. 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
were separated by use of optimum 2D TLC systems.
Authors:Alka Horvat, Zvonimir Šoljić, and Mirela Debelić
Two-dimensional thin-layer chromatography (2D TLC) has been used as a screening method for detection of the metal ions Al
, Sn(IV), and Fe
in honey. Different mobile phases and visualization reagents were tested. The best separation was achieved on precoated microcrystalline cellulose TLC plates with acetonitrile-HCl-water, 73 + 15 + 12 (
), as mobile phase in the direction
, and 2-pentanol-acetonitrile-HCl, 30 + 20 + 50 (
), in the direction
. A mixture of quercetin, dimethylglyoxime, and NH
was used for visualization of the chromatographic spots. This reagent mixture produced intensely colored spots for all the ions tested and enabled their identification at concentrations ranging from 50 to 1000 ng per spot.The method was used to identify metal ions in honey samples, dissolved in 2 mol L
hydrochloric acid, after microwave digestion. Seven metal ions, Al
and Sn(IV), were identified.
A 2D-separation of 16 polyaromatic hydrocarbons (PAHs) according to the Environmental Protecting Agency (EPA) standard was introduced. Separation took place on a TLC RP-18 plate (Merck, 1.05559). In the first direction, the plate was developed twice using n-pentane at −20°C as the mobile phase. The mixture acetonitrile-methanol-acetone-water (12:8:3:3, v/v) was used for developing the plate in the second direction. Both developments were carried out over a distance of 43 mm. Further on in this publication, a specific and very sensitive indication method for benzo[a]pyrene and perylene was presented. The method can detect these hazardous compounds even in complicated PAH mixtures. These compounds can be quantified by a simple chemiluminescent reaction with a limit of detection (LOD) of 48 pg per band for perylene and 95 pg per band for benzo[a]pyrene. Although these compounds were separated from all other PAHs in the standard, a separation of both compounds was not possible from one another. The method is suitable for tracing benzo[a]pyrene and/or perylene. The proposed chemiluminescence screening test on PAHs is extremely sensitive but may indicate a false positive result for benzo[a]pyrene.
Authors:Lukasz Cieśla, Anna Petruczynik, Michał Hajnos, Anna Bogucka-Kocka, and Monika Waksmundzka-Hajnos
Coumarins are interesting group of natural compounds, because of their biological and pharmacological activity, and are widely investigated. They are normally found in complex mixtures, e.g. plant extracts, and are difficult to separate in one chromatographic run. Mixtures of coumarins have been separated by two-dimensional thin-layer chromatography on CN-silica plates by use of aqueous and nonaqueous mobile phases. Complete separation was also achieved by use of graft thin-layer chromatography on connected layers — silica with RP-18W or CN-silica with silica. The systems characterized by the best efficiency and selectivity were used for separation of coumarin fractions from extracts of selected
plants. These orthogonal systems were used for quantitative analysis of selected coumarins. The results obtained show two dimensional thin-layer chromatography is useful tool for quantification of some furanocoumarins in plant extracts. The best results were obtained on connected silica and octadecyl silica layers.
Eighteen pesticides have been separated by two-dimensional thin-layer chromatography on a moderate polarity CN-modified silica gel. CN-silica is widely used as a adsorbent in both normal- and reversed-phase chromatography. Large selectivity differences are obtained by combination of both NP and RP modes on cyanopropyl-bonded polar adsorbents. The greatest spread of points was obtained by combining nonaqueous normal-phase mobile phases (tetrahydrofuran or ethyl acetate in
-heptane) and aqueous reversed phases (a polar solvent (methanol or acetonitrile) in water), both on thin layers of cyanopropyl-bonded polar adsorbent. Correlations of
values in NP and RP systems were used for practical separation of a mixture of eighteen pesticides by 2D TLC on this adsorbent. Plates were scanned and videoscanned to show the real pictures of the 2D TLC separation.
We present a two-dimensional (2D) planar chromatographic separation method for phytoestrogenic active compounds on RP-18 W (Merck, 1.14296) phase. It could be shown that an ethanolic extract of liquorice (Glycyrrhiza glabra) roots contains four phytoestrogenic active compounds. As solvent, in the first direction, the mix of hexane, ethyl acetate, and acetone (45:15:10, v/v) was used, and, in the second direction, that of acetone and water (15:10, v/v) was used. After separation, a modified yeast estrogen screen (YES) test was applied, using the yeast strain Saccharomyces cerevisiae BJ3505. The test strain (according to McDonnell) contains the estrogen receptor. Its activation by estrogen active compounds is measured by inducing the reporter gene lacZ which encodes the enzyme β-galactosidase. This enzyme activity is determined on plate by using the fluorescent substrate MUG (4-methylumbelliferyl-β-d-galactopyranoside). The enzyme can also hydrolyse X-β-Gal (5-bromo-4-chloro-3-indoxyl-β-d-galactopyranosid) into β-galactose and 5-bromo-4-chloro-3-indoxyl. The indoxyl compound is oxidized by oxygen forming the deep-blue dye 5,5β-dibromo-4,4β-dichloro-indigo which allows to detect phytoestrogenic activity more specific in the presence of native fluorescing compounds.
The selectivities of TLC systems have been compared by use of correlations between
values (by analogy with two-dimensional TLC). The greatest spread of spots, indicative of individual selectivity, was obtained for combination of non-aqueous mobile phases comprising a weakly polar diluent (heptane) and a polar modifier (tetrahydrofuran) on silica, and for aqueous mobile phases comprising a polar solvent (methanol) in water on water-wettable octadecyl silica (RP-18W). A good spread of spots was also obtained for pairs of normal-phase systems with hepatane-ethyl acetate mobile phases and reversed-phase systems with water-dioxane mobile phases, both on polar cyanopropyl-bonded layers. The correlation of
values in normal- and reversed-phase systems were used for practical separation of a mixture of ten urea herbicides by two-dimensional thin-layer chromatography on cyanopropyl-bonded polar adsorbents and by use of a layer comprising two zones — a narrow zone of silica adjacent to a wide zone of octadecyl silica (Multi-K SC5 plates). The plates were videoscanned to give real pictures of TLC chromatograms.
Authors:Marica Medić-Šarić, Ivona Jasprica, Ana Mornar, Asja Smolčić-Bubalo, and Petra Golja
Flavonoids are polyphenolic compounds present in a wide variety of plants. They are benzo-
-pyrone derivatives, which resemble coumarin. Flavonoids (with other polyphenols, e.g. phenolic acids) are believed to have a variety of physiological activity. One of the most concentrated sources of those compounds is propolis, a resinous substance collected by the honeybees from various plant sources. Its composition, and thus its content of pharmacologically active compounds, depends on geographic origin.We have developed a new TLC method suitable for analysis of complex mixtures such as propolis. Two-dimensional TLC with densitometric evaluation seemed an appropriate method for rapid screening of active compounds and quantifying the concentration of flavonoid aglycones and phenolic acids present in propolis extracts. After developing the method we tested it by analyzing and comparing the composition of three raw propolis samples from different geographic regions of Croatia.To establish the
values, standard solutions of nine flavonoids and six phenolic acids were first applied to the chromatographic plate as 10-mm bands. Plates were developed in vertical glass chambers previously saturated with the mobile phases —
-hexane-ethyl acetate-glacial acetic acid, 31 + 14 + 5 (
), (mobile phase 1) or chloroform-methanol-formic acid, 44 + 3.5 + 2.5 (
), (mobile phase 2). After drying, bands were visualized under short-and long-wavelength UV light; the plates were then sprayed with 1% AlCl
ethanol solution and viewed again under long-wavelength UV light.
values were calculated. All standards were chromatographed again in groups of 3 or 4 together with a propolis extract. First, plates were developed with mobile phase 1 (or 2), the mobile phase was evaporated, standard solutions were applied again, and the plate was rotated through 90° and chromatographed again with mobile phase 2 (or 1). The presence (or absence) of all standards was determined according to their
values and fluorescence colors. A better combination of mobile phases was chosen, the amounts of standards present were adjusted, and the standards were again individually chromatographed with the propolis extract. Quantitative evaluation was performed with Camag Reprostar 3 densitometer.Two-dimensional TLC was shown to be a highly suitable method for the task. We proved the method could be used for analysis of different propolis samples to identify and quantify the main pharmacologically active compounds.