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:Anna Petruczynik, Adrianna Karczmarz, and Monika Waksmundzka-Hajnos
The retention behavior of selected alkaloids on C18 and CN stationary phases in different eluent systems — mixtures of methanol and water with addition of buffers, acetic acid, ammonia, 1-octanesulfonic acid sodium salt, or diethylamine — has been determined. The separation of weak organic bases such as alkaloids is difficult because of their partial ionization in aqueous solutions. Improvement of separation selectivity, spot shapes, and system efficiency by use of special development methods: unidimensional multiple development (UMD), incremental multiple development (IMD), gradient multiple development (GMD), bivariant multiple development (BMD), and two-dimensional thin-layer chromatography (2D-TLC) was obtained. The effect of applied special development methods on chromatographic parameters obtained for investigated alkaloids in eluents of various composition on two layers was examined.
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.
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.
Authors:Tomasz Tuzimski, Anna Petruczynik, and Justyna Misiurek
In this study, authors propose a simple and cost-effective two-dimensional thin-layer chromatography (2D-TLC) method for the simultaneous determination of eleven standards alkaloids: allocryptopine (A), berberine (Be), boldine (Bo), chelidonine (Ch), glaucine (G), papaverine (Pa), emetine (E), columbamine (Col), magnoflorine (M), palmatine (Pal), and coptisine (Cop). Separation of the alkaloid mixture was achieved by 2D-TLC using an aqueous mobile phase (RP) in the first dimension (80% methanol + water + 0.05 mL−1 diethylamine) and a normal phase (NP) in the second dimension (18% methanol, 18% acetone in 63% diisopropyl ether containing 1% ammonia, v/v) on bilayer Multi-K CS5 plates. The composition of the mobile phases was optimized in terms of retention, separation selectivity, spots symmetry, and system efficiency. The procedure was evaluated in terms of natural samples analysis. Magnoflorine and berberine were identified in Thalictrum foetidum root extract. Additionally, the alkaloids in the extract sample were confirmed by high-performance liquid chromatography—diode-array detection method.
A simple and inexpensive two-dimensional thin-layer chromatography (2D-TLC) method was applied with mobile phase containing ionic liquid (IL) for the separation of ten components of a mixture of isoquinoline alkaloids such as allocryptopine (A), berberine (Be), boldine (Bo), chelidonine (Ch), papaverine (Pa), emetine (E), columbamine (Col), magnoflorine (M), palmatine (Pal), and coptisine (Cop). Separation of the alkaloid mixture was achieved by 2D-TLC using an aqueous mobile phase (RP) in the first dimension (80% methanol—water–0.05 M L−1 diethylamine) and a normal phase (NP) in the second dimension (75% methanol [MeOH], 24.75% ethyl methyl ketone–0.25% IL (1-butyl-3-methylimidazolium tetrafluoroborate) (v/v) on bilayer Multi-K CS5 plates. The procedure was evaluated in terms of natural samples analysis. Coptisine, palmatine, and berberine were identified in Rhizoma Coptidis (Huang Lian) extract. Additionally, the alkaloids in the extract sample were confirmed by high-performance liquid chromatography–diode-array detection (HPLC—DAD) method with application of a Synergi Polar RP column and a mobile phase consisting of a mixture of 25% acetonitrile (MeCN), 0.25% IL (1-butyl-3-methylimidazolium tetrafluoroborate), and 74.75% water (v/v). The composition of the mobile phases was optimized in terms of retention, separation selectivity, spots symmetry, and system efficiency. Limits of detection (LODs) were from 0.10 and 0.13 μg mL−1 and limits of quantification (LOQs) between 0.31 and 0.41 μg mL−1.
Authors:Mirosław Hawrył, Małgorzata Niemiec, and Monika Waksmundzka-Hajnos
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.