Paweł K. Zarzycki

Microfluidic Paper-Based Analytical Devices (μPADs) and Micro Total Analysis Systems (μTAS): Development, Applications and Future Trends

Microfluidic paper-based analytical devices and micro total analysis systems are relatively new group of analytical tools, capable of analyzing complex biochemical samples containing macromolecules, proteins, nucleic acids, toxins, cells or pathogens. Within one analytical run, fluidic manipulations like transportation, sorting, mixing or separation are available. Recently, microfluidic devices are a subject of extensive research, mostly for fast and non-expensive biochemical analysis but also for screening of medical samples and forensic diagnostics. They are used for neurotransmitter detection, cancer diagnosis and treatment, cell and tissue culture growth and amplification, drug discovery and determination, detection and identification of microorganisms. This review summarizes development history, basic fabrication methods, applications and also future development trends for production of such devices.

Simple horizontal chamber for thermostated micro-thin-layer chromatography

The aim of presented work is to describe simple, fast and robust temperature-controlled system for non-forced-flow micro-planar chromatography. With this separation system the micro-TLC plates can be developed in horizontal position under temperature gradient or non-gradient as well as saturated or unsaturated chamber conditions using low amount of mobile phase ranging from 0.3 to 1.0 mL. The device may work at wide range of temperatures from -20 to 80 degrees C. Under such conditions the plate temperature equilibration can be obtained within 5-12 min and a typical non-forced flow run can be finished within short period of time ranging from 5 to 20 min. It has been revealed that micro-plate is capable to separate more than 10 spots in one direction or up to 180 spots per plate for two-dimensional and multi-development runs. Particularly, fast and efficient separation of number of analytes including fullerenes, cyclodextrins and steroids as well as complex samples obtained from natural products and pharmaceutical formulations was demonstrated. Moreover, the application of thermostated micro-planar chromatography for the retention and quantification studies is also discussed.

Effect of temperature on separation of norgestrel enantiomers by high-performance liquid chromatography

The influence of mobile phase composition, concentration of beta-cyclodextrin and temperature on the high-performance liquid chromatographic separation of norgestrel was studied. In studies of the effect of temperature on the enantioselectivity of (+/-)-norgestrel, acetonitrile-water (25:75, v/v) modified by the addition of beta-cyclodextrin (14 mM) was applied as the mobile phase. Enantiomers were detected using UV detection at 240 nm. The capacity factors were measured over a wide range of column temperatures from -5 to 70 degrees C.

Application of β-cyclodextrin for the analysis of estrogenic steroids in human urine by high-performance liquid chromatography

SummaryA procedure is described for simultaneous determination of estriol, estrone and 17β-estradiol in human urine. After acid hydrolysis of the sulphate conjugates, the estrogens were extracted into diethyl ether. The ether extracts were concentrated and applied directly to an HPLC column. Using a 25 cm C-18 column and acetonitrile-water modified by the addition of β-cyclodextrin as mobile phase, the separation of estriol, estradiol and estrone was achieved within 20 minutes. The extraction of estrogens from the biological matrix is excellent. Estrogens were detected using a UV-detector (280 nm) or a spectrofluorimetric detector (λexc=280nm, λem=312 nm). The latter detection system has been used for the determination of estrogens in the urine of non-pregnant women. The procedure is simple and can be used in clinical practice.

Chromatographic behaviour of selected steroids and their inclusion complexes with β-cyclodextrin on octadecylsilica stationary phases with different carbon loads

Retention and separation studies of selected estrogens, progestogens and their inclusion complexes with beta-cyclodextrin were conducted using two C18 HPLC columns with different carbon loads. The difference in carbon load between investigated octadecylsilica packing materials was about 50%. The mobile phases were composed of a 30% v/v acetonitrile-water mixture without and with addition of beta-cyclodextrin at a concentration of 12 mM. The experimental data revealed that retention of the steroids was significantly reduced on the column with the lower carbon load. Moreover, it was found that this column offers better separation power and shorter analysis time at the temperatures studied. However, the calculated values of the retention factor ratios (k0(mMCD))/k(12mMCD)) of the steroids were similar for both columns investigated. This observation suggests that the stationary phase structure appears to have little effect on the formation of host-guest complexes if the complexation process is localised to the chromatographic mobile phase. From a practical point of view, when the mobile phase is modified with beta-cyclodextrin, the separation of the steroids is strongly influenced by temperature. The best chromatographic conditions were determined for the separation of multicomponent samples on the column with lower carbon load. A possible retention mechanism for components of interest in the presence of macrocyclic additives is discussed.

Retention and separation studies of cholesterol and bile acids using thermostated thin-layer chromatography.

The influence of temperature on retention and separation of cholesterol and bile acids, using reversed-phase thin-layer chromatography, was studied. As mobile phases methanol-water mixtures of various compositions were used. Chromatographic experiments were performed using vapor-saturated chambers at temperatures ranging from 5 to 60 degrees C. A linear relationship between R(M) values and temperature (1/T) as well as mobile phase composition was observed. The elution order of steroids under the conditions investigated was discussed. Each chromatogram was evaluated using simple optimization parameters and the best chromatographic conditions for the separation of multicomponent samples were chosen.

Low-parachor solvents extraction and thermostated micro-thin-layer chromatography separation for fast screening and classification of spirulina from pharmaceutical formulations and food samples.

The goal of this paper is to demonstrate the separation and detection capability of eco-friendly micro-TLC technique for the classification of spirulina and selected herbs from pharmaceutical and food products. Target compounds were extracted using relatively low-parachor liquids. A number of the spirulina samples which originated from pharmaceutical formulations and food products, were isolated using a simple one step extraction with small volume of methanol, acetone or tetrahydrofuran. Herb samples rich in chlorophyll dyes were analyzed as reference materials. Quantitative data derived from micro-plates under visible light conditions and after iodine staining were explored using chemometrics tools including cluster analysis and principal components analysis. Using this method we could easily distinguish genuine spirulina and non-spirulina samples as well as fresh from expired commercial products and furthermore, we could identify some biodegradation peaks appearing on micro-TLC profiles. This methodology can be applied as a fast screening or fingerprinting tool for the classification of genuine spirulina and herb samples and in particular may be used commercially for the rapid quality control screening of products. Furthermore, this approach allows low-cost fractionation of target substances including cyanobacteria pigments in raw biological or environmental samples for preliminary chemotaxonomic investigations. Due to the low consumption of the mobile phase (usually less than 1 mL per run), this method can be considered as environmentally friendly analytical tool, which may be an alternative for fingerprinting protocols based on HPLC machines and simple separation systems involving planar micro-fluidic or micro-chip devices.

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.

Optimization of TLC detection by phosphomolybdic acid staining for robust quantification of cholesterol and bile acids

In this paper we describe a robust and sensitive detection procedure for cholesterol and selected bile acids (cholic acid, lithocholic acid, and taurodeoxycholic acid sodium salt) using the common derivati-zation reagent phosphomolybdic acid (PMA). Visualization conditions were studied and optimized for steroids separated on glass TLC and HPTLC plates coated with silica gel (K60WF254S) and octadecylsilane (RP-18W) stationary phases. Spot intensities on the plates were quantified after spraying with PMA in methanol (10% w/v) and heating at temperatures from 40 to 120°C for times ranging from 2 to 40 min. The best conditions for high signal intensity were determined by using 3D temperature (X)-time (Y)-analytical signal (Z) maps generated from the raw experimental data. In contrast with the number of “universal procedures” described in the literature our study indicated that for robust and sensitive quantification of our components of interest heating should be performed at relatively low temperatures (below 100°C) and for heating times in excess of 10 min. Particularly robust and sensitive detection of steroids separated on glass plates coated with both stationary phases was observed for temperatures ranging between 50 and 80°C and heating for at least 20 min in a simple gravity convection oven.

Interactions between native cyclodextrins and n-alcohols studied using thermostated thin-layer chromatography

Abstract Using thermostated thin-layer chromatography, the retention behavior of α-, β- and γ-cyclodextrin on a C18 stationary phase was studied. As mobile phases, a homologous series of n-alcohols, from ethanol to pentanol, and their mixtures with water were examined. Chromatographic experiments were performed either at a constant temperature (30°C) and over a wide range of binary mixtures (0–100%, v/v), for ethanol and propanol, as well as at fixed mobile phase composition and different temperatures from 5°C to 60°C. Using isoelution binary mobile phases, the effect of temperature on retention of cyclodextrins was examined. Results were compared with chromatographic retention data from previously reported work in which methanol–water binary phases were used. From linear Van ‘t Hoff plots thermodynamic parameters such as the change of enthalpy (ΔH0) and the change of entropy (ΔS0) were estimated. In each case the sign of the calculated parameters is negative. Nonlinearities of Van ‘t Hoff plots were observed when propanol or butanol was used as a component of binary mobile phase.