Authors:Monika Waksmundzka-Hajnos and Grzegorz Jóźwiak
A strategy has been established for preparative-layer chromatography of the alkaloid fraction from
. An extract obtained by maceration and percolation of the milled plant material with 0.5 m
aqueous acetic acid was re-dissolved in methanol and fractionated by column chromatography on alumina. Two of the fractions obtained, containing medium polarity alkaloids and strongly polar alkaloids, respectively, were used in the preparative-layer chromatography experiments. Preliminary work focused on investigation of overloading, methods of sampling, effect of layer thickness on band resolution, and the effect of derivatization on the location of separated bands. Another objective of the work was to investigate the use of unidimensional multiple development (UMD) and incremental multiple development (IMD) for resolution of the alkaloids found in
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:Monika Waksmundzka-Hajnos and Anna Petruczynik
The retention behavior of twenty-four alkaloids has been investigated in normal-phase systems on thin layers of aminopropylsilica and bare silica developed with a variety of binary mobile phases prepared from n-hexane and polar modifiers – 2-propanol, ethyl acetate, methyl ethyl ketone, tetrahydrofuran, dioxane – in different concentrations. The retention factors obtained were fitted to the logarithmic Snyder–Soczewiński equation; the results are presented as the equation parameters and the statistical coefficients which prove the goodness of fit of the results with the displacement theory of retention.
The retention behavior of the alkaloids was also investigated in reversed-phase systems with buffered aqueous–acetonitrile mobile phases (pH 9.15) and the cyanopropyl- and octadecylsilica as stationary phases. Retention factors obtained for use of different concentrations of acetonitrile in the mobile phase were fitted to the semi-logarithmic equation or to quadratic polynomial equation selected for RP systems and are also presented as equation parameters and statistical coefficients which prove the goodness of fit of the results obtained with the theories derived for RP systems.
Separation selectivity was presented as the RF spectrum for the aminopropyl phase, comparing the separation selectivity obtained by use of different mobile phases, and as RF1 vs RF2 correlation diagrams for different systems, enabling selection of the most selective systems for separation of particular pairs or groups of alkaloids. Such correlation diagrams are presented for the aminopropyl phase and silica with four non-aqueous mobile phases and also for CN-silica and C18 phases, and for CN-silica and diol phases with aqueous mobile phases.
Authors:Monika Waksmundzka-Hajnos, Maria Gadzikowska, and Michał Hajnos
L. quaternary alkaloid fraction has been used as a model mixture to investigate the effect of the procedure used on the preparative separation of its components. The steps optimized wereThe mode of application of the starting band to the layerThe effect on the separation of the bands of the number of developments in unidimensional multiple development (UMD)The effect of concentration overloading on the separation of the bands; andThe effect of the volume of solution introduced to the adsorbent layer on the resolution of neighboring bands.
Authors:Anna Petruczynik, Monika Waksmundzka-Hajnos, and Michał Hajnos
Alkaloid standards have been chromatographed on silica layers with a variety of aqueous and nonaqueous mobile phases. The effect on retention, efficiency, and peak symmetry of pH, anionic ionpairs, DEA, and ammonia in aqueous mobile phases was examined. The effect of the concentration of ion-suppressing reagent — diethylamine — on retention, separation efficiency, and peak shape in normal-phase systems was also studied. Systems with the best selectivity and efficiency were used to separate alkaloid standards mixtures and plant extracts by 2D TLC.
Authors:Łukasz Cieśla, Michał Hajnos, and Monika Waksmundzka-Hajnos
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-H2O-25% aqueous NH3 (55:35:9:1, ν/ν/ν/ν) and redeveloped with MeOH-AcOEt-H2O-HCOOH (10:90:26:22, ν/ν/ν/ν) 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.
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, Daniel Cichocki, and Monika Waksmundzka-Hajnos
Psychotropic drugs have been chromatographed on RP-18-HPTLC plates with mobile phases containing water, an organic modifier (methanol, dioxane, acetone, acetonitrile or tetrahydrofuran), and ion-pair reagents or ammonia.
was measured for different concentrations of organic modifier. Relationships between solute retention and modifier concentration were described by the
equations. The equations were used to determine
by the extrapolation method. Calculated values of
were correlated with log
values for the drugs, estimated by use of software. Significant correlations between the intercept (
) and slope (
) of the linear equations were also calculated. Chromatographic systems for which the
values obtained were close to calculated log
values can be used for determination of the lipophilicity of basic drugs.
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.