Anna Petruczynik

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.

Comparison of chromatographic properties of cyanopropyl-, diol- and aminopropyl- polar-bonded stationary phases by the retention of model compounds in normal-phase liquid chromatography systems.

Polar-bonded stationary phases, such as CN-, diol- and NH2-silica, have been characterised by the retention of model solutes (phenols, aromatic amines and quinoline bases) in normal-phase systems using n-heptane--polar modifier (2-propanol, tetrahydrofuran or dioxane) mixtures as eluents. The selectivity of separation for the particular groups of substances has been analysed by the log kI versus log kII relationships for CN- and diol, CN- and NH2- and NH2- and diol phases in examined eluent systems by the plotting of correlation lines. The values of regression coefficient r indicate either the similarity of the retention mechanisms of model solutes in some examined systems where r>0.9, or differences among various systems where r<<0.9. The values of slopes of correlation lines show the selectivity of separation for particular group of compounds. The selectivity of separation has also been characterised by deltalog k values. The effect of modifier (2-propanol, tetrahydrofuran and dioxane) on selectivity of model solutes on these phases has also been discussed.

Effect of Ionic Liquid Additives to Mobile Phase on Separation and System Efficiency for HPLC of Selected Alkaloids on Different Stationary Phases

The silica-based stationary phases with favorable physical characteristics are the most popular in liquid chromatography. However, there are several problems with silica-based materials: severe peak tailing in the chromatography of basic compounds, non-reproducibility for the same chemistry columns, and limited pH stability. Ionic liquids (ILs) as mobile phase components can reduce peak tailing by masking residual free silanol groups. The chromatographic behavior of some alkaloids from different classes was studied on C18, phenyl, and pentafluorophenyl columns with different kinds and concentrations of ionic liquids as additives to aqueous mobile phases. Ionic liquids with different alkyl substituents on different cations or with different counterions as eluent additives were investigated. The addition of ionic liquids has great effects on the separation of alkaloids: decrease in band tailing, increase in system efficiency, and improved resolution. The retention, separation selectivity, and sequence of alkaloid elution were different when using eluents containing various ILs. The increase of IL concentration caused an increase in silanol blocking, thus conducted to decrease the interaction between alkaloid cations and free silanol groups, and caused a decrease of alkaloids retention, improvement of peak symmetry, and increase of theoretical plate number in most cases. The effect of ILs on stationary phases with different properties was also examined. The different properties of stationary phases resulted in differences in analyte retention, separation selectivity, peak shape, and system efficiency. The best shape of peaks and the highest theoretical plate number for most investigated alkaloids in mobile phases containing IL was obtained on pentafluorophenyl (PFP) phase.

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.

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.

Two-Dimensional Thin-Layer Chromatography of Structural Analogs. Part II. Method for Quantitative Analysis of Selected Coumarins in Plant Material

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 Apiaceae 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.

Retention of some organic electrolytes in ion-pair reversed-phase high-performance liquid and reversed-phase high-performance thin-layer chromatographic systems

Abstract Cetyltrimethylammonium bromide (cetrimide) and tetrabutylammonium chloride were employed as ion-pairing reagents in reversed-phase ion-pair chromatography. The optimization of the retention and selectivity for some N-phenylamides of benzoylacetic acid was carried out by changing the content of the organic modifier (methanol) and the concentration of the ion-pairing reagent in the mobile phase.

The effect of chromatographic conditions on the separation of selected alkaloids on silica layers

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.

Analysis of alkaloids from different chemical groups by different liquid chromatography methods

AbstractAlkaloids are biologically active compounds widely used as pharmaceuticals and synthesised as secondary methabolites in plants. Many of these compounds are strongly toxic. Therefore, they are often subject of scientific interests and analysis. Since alkaloids — basic compounds appear in aqueous solutions as ionized and unionized forms, they are difficult for chromatographic separation for peak tailing, poor systems efficiency, poor separation and poor column-to-column reproducibility. For this reason it is necessity searching of more suitable chromatographic systems for analysis of the compounds.In this article we present an overview on the separation of selected alkaloids from different chemical groups by liquid chromatography thus indicating the range of useful methods now available for alkaloid analysis. Different selectivity, system efficiency and peaks shape may be achieved in different LC methods separations by use of alternative stationary phases: silica, alumina, chemically bonded stationary phases, cation exchange phases, or by varying nonaqueous or aqueous mobile phase (containing different modifier, different buffers at different pH, ion-pairing or silanol blocker reagents). Developments in TLC (NP and RP systems), HPLC (NP, RP, HILIC, ion-exchange) are presented and the advantages of each method for alkaloids analysis are discussed.

The Retention of Some Organic Acids in Ion-Pair HPLC Systems

Abstract The retention behaviour of some organic acids (N-phenylamides of benzoylacetic acid, phenolic acids and analgesic drugs) as model substances was investigated in reversed phase systems consisting of octadecyl silica (ODS) as a column packing material eluted with the buffer-methanol mixtures containing low concentrations of cetyltrimethylammonium bromide (cetrimide), tetrabutylammonium chloride (TBA-C1), tetraethylammonium chloride (TEA-C1) and di(2-ethylhexyl) orthophosphoric acid (HDEHP). The chain length of the n-alkyl group of the ion-pair reagent and the content of a modifir in the eluent contribute to rtention. Correlation between log k and log P and biological activity of N-phenylamides was analysed.