Elżbieta Włodarczyk

Application of micro-thin-layer chromatography as a simple fractionation tool for fast screening of raw extracts derived from complex biological, pharmaceutical and environmental samples.

The main goal of present paper is to demonstrate the separation and detection capability of micro-TLC technique involving simple one step liquid extraction protocols of complex materials without multi-steps sample pre-purification. In the present studies target components (cyanobacteria pigments, lipids and fullerenes) were isolated from heavy loading complex matrices including spirulina dried cells, birds feathers and fatty oils as well as soot samples derived from biomass fuel and fossils-fired home heating systems. In each case isocratic separation protocol involving less that 1 mL of one component or binary mixture mobile phases can be completed within time of 5-8 min. Sensitive detection of components of interest was performed via fluorescence or staining techniques using iodine or phosphomolybdic acid. Described methodology can be applied for fast fractionation or screening of whole range of target substances as well as chemo-taxonomic studies and fingerprinting of complex mixtures, which are present in raw biological or environmental samples.

Temperature-controlled micro-TLC: a versatile green chemistry and fast analytical tool for separation and preliminary screening of steroids fraction from biological and environmental samples.

This paper is a continuation of our previous research focusing on development of micro-TLC methodology under temperature-controlled conditions. The main goal of present paper is to demonstrate separation and detection capability of micro-TLC technique involving simple analytical protocols without multi-steps sample pre-purification. One of the advantages of planar chromatography over its column counterpart is that each TLC run can be performed using non-previously used stationary phase. Therefore, it is possible to fractionate or separate complex samples characterized by heavy biological matrix loading. In present studies components of interest, mainly steroids, were isolated from biological samples like fish bile using single pre-treatment steps involving direct organic liquid extraction and/or deproteinization by freeze-drying method. Low-molecular mass compounds with polarity ranging from estetrol to progesterone derived from the environmental samples (lake water, untreated and treated sewage waters) were concentrated using optimized solid-phase extraction (SPE). Specific bands patterns for samples derived from surface water of the Middle Pomerania in northern part of Poland can be easily observed on obtained micro-TLC chromatograms. This approach can be useful as simple and non-expensive complementary method for fast control and screening of treated sewage water discharged by the municipal wastewater treatment plants. Moreover, our experimental results show the potential of micro-TLC as an efficient tool for retention measurements of a wide range of steroids under reversed-phase (RP) chromatographic conditions. These data can be used for further optimalization of SPE or HPLC systems working under RP conditions. Furthermore, we also demonstrated that micro-TLC based analytical approach can be applied as an effective method for the internal standard (IS) substance search. Generally, described methodology can be applied for fast fractionation or screening of the whole range of target substances as well as chemo-taxonomic studies and fingerprinting of complex mixtures, which are present in biological or environmental samples. Due to low consumption of eluent (usually 0.3-1mL/run) mainly composed of water-alcohol binary mixtures, this method can be considered as environmentally friendly and green chemistry focused analytical tool, supplementary to analytical protocols involving column chromatography or planar micro-fluidic devices.

Determination of endocrine disrupting compounds using temperature-dependent inclusion chromatography: I. Optimization of separation protocol

In the present work we optimised the separation of battery of key UV non-transparent low-molecular-mass compounds having possible endocrine disrupting compounds (EDCs) activity or which may be used as the endocrine effect biomarkers. Simple optimization strategy was based on strong temperature effect that is driven by electrostatic interactions between macrocyclic mobile phase additives like cyclodextrins and eluted components of interest under C18 stationary phase and acetonitrile/water mobile phase conditions. Particularly, the effect of temperature involving native beta-cyclodextrin and its hydroxypropyl derivative to improve separation of number of natural (d-equilenin, equilin, estetrol, estriol, estrone, 17beta-estradiol, 17alpha-hydroxyprogesterone, 20alpha-hydroxyprogesterone, cortisol, cortisone, progesterone, testosterone, tetrahydrocortisol and tetrahydrocortisone) and artificial steroids (ethynylestradiol, norgestrel isomers, medroxyprogesterone, mestranol, methyltestosterone, norethindrone, 17alpha-estradiol) as well as non-steroidal compounds (diethylstilbesterol, bisphenol A, 4-tert-butylphenol, dimethyl phthalate, dibutyl phthalate and dioctyl phthalate) was investigated. It has been found that successful isocratic separation of 27 chemicals can be achieved using acetonitrile/water eluents modified with beta-cyclodextrin or hydroxypropyl-beta-cyclodextrin at concentration of 10 mM and temperature of 47 degrees C. Separation protocol is simple, reliable, direct and non-radioactive and may be easily adapted for rapid separation and quantification of wide range of given steroids and related EDCs in environmental samples, particularly those that are characterised by unstable biological matrix and components of interest load.

Determination of endocrine disrupting compounds using temperature-dependent inclusion chromatography: II. Fast screening of free steroids and related low-molecular-mass compounds fraction in the environmental samples derived from surface waters, treated and untreated sewage waters as well as activated sludge material

In the present work solid-phase extraction protocol based on C18 tubes and organic water washing solvents as well as isocratic HPLC procedure focused on quantification of free steroids and related low-molecular-mass endocrine disrupting compounds (EDCs), characterized by different polarity varied from estetrol to progesterone, were studied. Described separation method involves temperature-dependent inclusion chromatography with mobile phase modified with beta-cyclodextrin. Using such analytical approach the environmental samples derived from Baltic Sea, selected lakes and rivers of the Middle Pomerania in northern part of Poland as well as untreated and treated sewage water from municipal sewage treatment plant near Koszalin were analyzed. Moreover, some preliminary data concerning estriol, testosterone and equilin biodegradation involving activated sludge material were reported. Cluster and principal components analysis of the acquired data sets confirms a high separation and quantification throughput of the solid-phase extraction and isocratic HPLC protocols presented. The method can be useful for simple and rapid classification of the environmental samples characterized by different sources of EDCs loading. The results of this work extend the utility of temperature-dependent inclusion chromatography as an inexpensive, efficient and accurate analytical tool appropriate for characterisation and quantification of complex environmental samples.

Micro-TLC Approach for Fast Screening of Environmental Samples Derived from Surface and Sewage Waters

In this work we demonstrated analytical capability of micro-planar (micro-TLC) technique comprising one and two-dimensional (2D) separation modes to generate fingerprints of environmental samples originated from sewage and ecosystems waters. We showed that elaborated separation and detection protocols are complementary to previously invented HPLC method based on temperature-dependent inclusion chromatography and UV-DAD detection. Presented 1D and 2D micro-TLC chromatograms of SPE (solid-phase extraction) extracts were optimized for fast and low-cost screening of water samples collected from lakes and rivers located in the area of Middle Pomerania in northern part of Poland. Moreover, we studied highly organic compounds loaded in the treated and untreated sewage waters obtained from municipal wastewater treatment plant “Jamno” near Koszalin City (Poland). Analyzed environmental samples contained number of substances characterized by polarity range from estetrol to progesterone as well as chlorophyll-related dyes previously isolated and pre-purified by simple SPE protocol involving C18 cartridges. Optimization of micro-TLC separation and quantification protocols of such samples were discussed from the practical point of view using simple separation efficiency criteria including total peaks number, log(product ΔhRF), signal intensity and peak asymmetry. Outcomes of the presented analytical approach, especially using detection involving direct fluorescence (UV366/Vis) and phosphomolybdic acid (PMA) visualization are compared with UV-DAD HPLC-generated data reported previously. Chemometric investigation based on principal components analysis revealed that SPE extracts separated by micro-TLC and detected under fluorescence and PMA visualization modes can be used for robust sample fingerprinting even after long-term storage of the extracts (up to 4xa0years) at subambient temperature (−20xa0°C). Such approach allows characterization of wide range of sample components that are present in given extract in high and middle concentration range. Due to protocol simplicity and low cost of analysis this method can be useful for preliminary sample screening.

Improved Detection of Ergosterol, Stigmasterol, and Selected Steroids on Silica Coated TLC Plates using Phosphomolybdic Acid Staining

Abstract In this work, improved detection of selected steroids on silica coated TLC plates using post‐run derivatisation with phosphomolybdic acid (PMA) was investigated. Particularly, spot visualization of ergosterol, stigmasterol, dihydrocholesterol, 4‐cholesten‐3‐one, cholecalciferol, and cholesterol acetate was studied. It has been found that the quantitative effect of PMA dyeing is strongly time and temperature dependent. Contrary to the data reported in literature, our experimental work revealed that the best conditions for robust detection of analytes investigated can be expected if the silica coated chromatographic plates are heated within a relatively low temperature region, between 40 and 80°C and for more than 20 minutes.

FINGERPRINTING OF SOOT DUST MATERIALS USING MICRO-TLC

Micro-planar chromatography with 45 mm developing distance involving reversed-phase HPTLC (RP18W) plates and simple n-hexane mobile phase was applied for fractionation of toluene extracts of soot dust materials. Raw samples for micro-TLC separation were derived from cold surfaces of biomass fuel and fossils-fired home heating ovens as well as trucks exhaust pipes. Discrimination of investigated objects was based on entire thin-layer chromatographic profiles and principal component analysis. Particularly, we demonstrated that such complex samples can be effectively fingerprinted and robustly classified using fluorescence (366 nm) and fluorescence quenching (254 nm) detection and multivariate data mining.

Unexpected differences between planar and column liquid chromatographic retention of 1-acenaphthenol enantiomers controlled by supramolecular interactions involving β-cyclodextrin at subambient temperatures

We report the results of experimental work focusing on host-guest supramolecular complex creation between macrocyclic compound (β-cyclodextrin) and 1-acenaphthenol enantiomers (racemic mixture) in liquid phase composed of 35% acetonitrile in water (v/v) at different temperatures ranging from 0 to 90xa0°C. Experimental setup involved several analytical protocols based on classical non-forced flow planar chromatography (RP-18 TLC plates), micro-TLC (RP-18 W HPTLC plates), column chromatography (HPLC with C-18 and C-30 stationary phases), as well as UV-Vis spectrophotometry and optical microscopy. It has been found that under various planar chromatographic conditions (stationary plates type, chamber shape and volume, development mode, and saturation) non-typical retention properties (extremely high retention) of 1-acenaphthenol at subambient temperatures can be observed. To our knowledge, reported experimental results are in opposition to currently described retention models based on column chromatographic investigation of host-guest complexes (where in case of strong interaction of given analyte with macrocyclic mobile phases additive, which itself is non strongly retarded by stationary phase—close to the retention of dead volume marker, the retention of target compounds is shortened at low temperatures). To explain this TLC phenomenon that may have in our opinion a number of practical applications, especially for selective high throughput separation involving microchromatographic and/or microfluidic devices as well fractionation and extraction protocols (using, e.g., bar extraction systems), several experiments were conducted focusing on (i) acenaphthenol chromatography under different instrumental conditions, (ii) cyclodextrin retention measured as analyte or mobile phase additive, (iii) plate development time under different mobile phases and temperature settings, (iv) various column chromatographic conditions including C-30 and two C-18 stationary phases, (v) UV-Vis spectrophotometry, and (vi) microscopy inspection of precipitated CD-acenaphthenol crystals. Analysis of collected data has revealed that the most probable reasons for TLC retention behavior of 1-acenaphthenol under β-cyclodextrin additive conditions can be associated with (i) solubility changes of created host-guest complex, (ii) kinetics of solid complex precipitation, and (iii) differences in analysis time between planar and column chromatography. Because precipitation phenomenon may have a massive impact on analytes quantification involving macrocycles as the mobile phase additives, our previously reported data concerning a number of low-molecular compounds (mainly steroids and non steroidal endocrine disrupting chemicals) using HPLC methodology based on binary mobile phases without and with β-cyclodextrin and its hydroxypropyl derivative were re-examined and results discussed. Considering these data and the whole data set reported presently, the enhanced model of chromatographic retention driven by host-guest interaction was proposed.

A preliminary study for the fast prototyping of simple electroplanar separation systems based on various natural polymers and planar chromatographic stationary phases

This experimental work deals with preliminary studies concerning combined planar electrophoresis—electrochromatography system involving stationary phases composed of various natural materials applied for the separation of selected colorants. The main goal of the presented experiment is to investigate the key parameters (applied voltage, electrolyte composition, pH) and separation protocol setup (stationary phase/connection strips type and geometry) enabling the fast prototyping of simple analytical systems for the fractionation and/or separation of low-molecular mass compounds. Four water-based electrolytes that are as simple as possible (without additional organic liquid components or modifiers), including boric acid 100 mM (pH = 4.3), 1:1 mixture of boric acid 90 mM and Tris base 90 mM (pH = 8.2), boric acid 100 mM titrated with NaOH 1 m (pH = 8.5), and formic acid 100 mM (pH = 2.4), were tested. As target analytes, two colorants, namely, methyl red and ponceau 4R, were selected. These dyes are commonly used as printing inks component or pH indicator (methyl red) and food products colorant (ponceau 4R). Electroseparation experiments were conducted using commercially available, temperature non-controlled, open-air electrophoresis box equipped with homemade support for the positioning of active separation layers and electrodes connection strips. Thirteen types of separation layers (working zone = 20 × 100 mm; total length with connection strips = 20 cm) including cellulose-based polymers (filtrating paper, office paper, chromatography paper, and two Japanese papers for aircraft paper models), potato starch on cellulose support, common thin-layer chromatography—high-performance thin-layer chromatography (TLC—HPTLC) glass-based plates coated with cellulose, silica gel 60W (wettable with water), silica gel RP-18W, and aluminum oxide as well as glass-based nutrient agar layers (0.5 and 1.0 mm, approximately) were investigated. Moreover, detailed preparation procedure for manufacturing starch layer on cellulose and agar glass plate supports is described. Conducted investigations have revealed substantial differences between the electrophoretic migration of target dyes within cellulose type layers and also in comparison to the remaining stationary phases studied. The best separation under the given analytical conditions (voltage applied ΔV = 500 V and run time 20 min) was observed for cellulose pre-coated TLC plate (Rs = 2.89) and starch layer on filtrating paper support (Rs = 2.26). Baseline separation of investigated dyes was observed for filtrating paper strip and agar 0.5-mm thick layer (Rs = 1.07 and 1.08, respectively). There was no electrophoretic mobility shift of the tested dyes on polyamide and RP-18W coated TLC—HPTLC plates, while dye short migration without separation was observed on the remaining layers. The obtained results, especially the recorded values of physicochemical parameters (the observed electric currents, migration distances, and peak resolution for different electrolytes, layer type, and thickness) create initial data set platform that can help in the further design of simple separation devices involving natural polymeric layers. Particularly, the described analytical protocol may be implemented for microfluidic paper-based analytical devices (μPADs) enabling fast and non-expensive separation of complex samples.

Toward the Understanding of Micro-TLC Behavior of Various Dyes on Silica and Cellulose Stationary Phases Using A Data Mining Approach

Planar chromatography and related techniques [micro-planar chromatography, micro-TLC, or paper-based microfluidic devices (μPADs)] present several advantages in analytical applications, such as simplicity, low cost of analysis, and the ability to work with raw complex samples without the involvement of time-consuming prepurification steps. By using commonly applied planar chromatographic systems and μPADs devices, stationary phases (silica and cellulose based), different solvent mixtures (methanol-water and dichloromethane-methanol), and proportions varying from 0 to 100% (v/v), micro-TLC migration profiles of several dyes described in terms of characteristic of chromatographic parameters (retardation factor, peak base width, and asymmetry factor) were investigated. Combining these results with some quantum mechanics calculated properties for each solute (dipole moment, polarizability), and by using the data mining approach, we modeled this overall chromatographic behavior in order to describe experimental data. With this approach, we were able to predict with reasonable confidence some chromatographic properties. This effort its crucial in order to (1) optimize solute elution, (2) increase mixture resolution, and (3) identify some molecular properties of analytes for designing simple micro-TLC. It is hoped that the presented nonhypothesis-driven data-mining approach can be helpful for understanding the chromatographic behavior of dyes on silica and cellulose adsorbents using the simplest mobile phases. This should be helpful for further designing the micro-TLC separation systems or μPADs quantification devices based on cellulose and related biopolymers and considering dye compounds as analytes for separation and sensing molecules.