Application of different modes of thin-layer chromatography and mass spectrometry for the separation and detection of large and small biomolecules

Biomolecules are widespread throughout the world. A biomolecule is any organic molecule produced by a living organism, including large polymeric molecules such as proteins, polysaccharides and nucleic acids. Many sample preparation techniques are used in biomolecule analysis; the method selected depends on the complexity of the sample, the nature of the matrix and the analytes, and the analytical technique available. This review covers the current state of knowledge on thin-layer chromatography and mass spectrometry for qualitative analysis of biomolecules. In the first part of the paper the reader will gain useful information to avoid some problems about performing various modes of thin-layer chromatography combined with mass spectrometry experiments and in the second part he will find useful information for application of these techniques for separation, detection, and qualitative investigation of structures and quantitative determination of biomolecules such as proteins, peptides, oligonucleotides, amino acids, DNA, RNA, and lipids.     

Application of HPLC-DAD for In Vitro Investigation of Acetylcholinesterase Inhibition Activity of Selected Isoquinoline Alkaloids from Sanguinaria canadensis Extracts

Isoquinoline alkaloids may have a wide range of pharmacological activities. Some of them have acetylcholinesterase activity inhibition. Nowadays, neurodegenerative disorders such as Alzheimer’s disease have become a serious public health problem. Searching for new effective compounds with inhibited acetylcholinesterase activity is one of the most significant challenges of modern scientific research. The aim of this study was the in vitro investigation of acetylcholinesterase activity inhibition of extracts obtained from Sanguinaria canadensis collected before, during and after flowering. The acetylcholinesterase activity inhibition of these extracts has not been previously tested. The aim was also to quantify selected alkaloids in the investigated extracts by high performance liquid chromatography (HPLC). The analyses of alkaloid content were performed using HPLC in reversed phase (RP) mode using Polar RP column and mobile phase containing acetonitrile, water and ionic liquid (IL). The acetylcholinesterase activity inhibition of the tested plant extracts and respective alkaloid standards were examined using high performance liquid chromatography with diode-array detector (HPLC-DAD) for the quantification of 5-thio-2-nitro-benzoic acid, which is the product of the reaction between the thiocholine (product of the hydrolysis of acetylthiocholine reaction) with Ellman reagent. The application of the HPLC method allowed for elimination of absorption of interfering components, for example, alkaloids such as sanguinarine and berberine. It is revealed that the HPLC method can be successfully used for the evaluation of the acetylcholinesterase inhibitory activity in samples such as plant extracts, especially those containing colored components adsorbing at wavelength in the range 405–412 nm. The acetylcholinesterase inhibition activity synergy of pairs of alkaloid standards and mixture of all investigated alkaloids was also determined. Most investigated alkaloids and all Sanguinaria canadensis extracts exhibited very high acetylcholinesterase activity inhibition. IC₅₀ values obtained for alkaloid standards were from 0.36 for berberine to 23.13 µg/mL for protopine and from 61.24 to 89.14 µg/mL for Sanguinaria canadensis extracts. Our investigations demonstrated that these plant extracts can be recommended for further in vivo experiments to confirm their acetylcholinesterase activity inhibition.     

A new procedure for separation of complex mixtures of pesticides by multidimensional planar chromatography

The objective of this communication is to report a new procedure for the separation of complex mixtures of pesticides by a combination of different modes of multidimensional planar chromatography. Complete separation of a mixture of pesticides was carried out by multidimensional planar chromatography on silica gel plates. The plates were videoscanned to show a real picture of the experimental results. Complete separation of all compounds was not possible by a single development on silica gel. The separation can be characterized as [PC x (nPC + PC + PC + PC)]. With the aid of this new procedure a complex mixture of fourteen compounds was separated on a 10 x 10 cm TLC plate.     

Determination of analytes in medical herbs extracts by SPE coupled with two-dimensional planar chromatography in combination with diode array scanning densitometry and HPLC-diode array detector

The purpose of this study is to demonstrate an application of 2-D high-performance planar chromatography-diode array detector (DAD) and HPLC-DAD after solid-phase extraction (SPE) for identification and quantitative analysis of pesticides (isoproturon, aziprotryne, hexazinone, flufenoxuron, methabenzthiazuron, procymidone, and α-cypermethrin) in Melissa officinalis L. (Labiatae) samples. The procedure described for the determination of compounds is inexpensive and can be applied to routine analysis of analytes in medical herbs' samples after preliminary cleanup and concentration by SPE. Average recoveries on C18 SPE cartridges of pesticides eluted with 5 mL tetrahydrofuran by the proposed HPLC-DAD method, before and after 2-D-high-performance planar chromatography separation of analytes from M. officinalis L. samples spiked with pesticide at a concentration level of 10 μg/g in plant material are presented. Method validation parameters for the quantification of pesticides by the proposed HPLC-DAD after SPE method are also presented.     

Separation of a Mixture of Eleven Alkaloids by 2D-TLC on Multi-K CS5 Plates and identification of Analytes in Thalictrum foetidum Root Extract by TLC and HPLC—DAD

JPC – Journal of Planar Chromatography – Modern TLC - In this study, authors propose a simple and cost-effective two-dimensional thin-layer chromatography (2D-TLC) method for the...     

Correlation of Retention Data of Pesticides in Normal- and Reversed-Phase Systems and Utilization of the Data for Separation of a Mixture of Ten Urea Herbicides by Two-Dimensional Thin-Layer Chromatography on Cyanopropyl-Bonded Polar Stationary Phase and on a Two-Adsorbent-Layer Multi-K SC5 Plate

JPC – Journal of Planar Chromatography – Modern TLC - The selectivities of TLC systems have been compared by use of correlations between RF(II) and RF(I) values (by analogy with...     

Use of a database of plots of pesticide retention (R F) against mobile-phase composition.Part II. TLC as a pilot technique for transferring retention data to HPLC, and use of the data for preliminary fractionation of a mixture of pesticides by micropreparative column chromatography

Summary Relationships betweenR _F values and mobile-phase composition have been determined for moderately polar pesticides in normal-phase systems (NP) of the type silica-non-polar diluent (heptane)-polar modifier (ethyl acetate, tetrahydrofuran, or dioxane) and in reversed-phase systems (RP) of the type octadecyl silica-water-polar modifier (acetonitrile, methanol, or tetrahydrofuran). These relationships constitute a retention database which has enabled choice of the optimum conditions for preparative column chromatographic separation of pesticides into fractions; these were then applied to a silica plate and chromatographed. The plate was videoscanned, furnishing a real picture of the plate showing complete separation of the pesticide fractions.     

Comparison of different HPLC systems for analysis of galantamine and lycorine in various species of Amaryllidaceae family

Retention parameters of galantamine and lycorine standards were determined on different columns, i.e., octadecyl silica, SM C18, and strong cation-exchange (SCX) columns with different aqueous mobile phases. Retention of alkaloids was investigated on C18, SM C18 columns with mobile phase containing 5% MeCN, 20% acetate buffer at pH 3.5, and 0.025 ML^?1 diethylamine (DEA), and on SCX column with mobile phase containing 8% MeCN and phosphate buffer at pH 2.5. Better results were also obtained in ion-exchange chromatographic system. On the basis of results obtained in different chromatographic systems, simple, rapid, and sensitive high-performance liquid chromatography methods were developed for determining lycorine and galantamine in plant extracts from various species belonging to Amaryllidaceae family. Extracts were prepared from various parts of plants collected at different times of the growing season.     

Application of SPE-HPLC-DAD and SPE-TLC-DAD to the determination of pesticides in real water samples

Planar chromatography with diode array scanning (TLC-DAD) and high-performance chromatography with diode array detection (HPLC-DAD) were used to screen water samples for pesticides. Pesticides were enriched from lake water samples by SPE on C18/SDB-1, C18, C18 Polar Plus and cyanopropyl (CN) cartridges. The recovery rates were high for all extraction materials except for all pesticides on CN cartridges, for which the values were lower. SPE was used not only for preconcentration of analytes but also for their fractionation. The analytes were eluted first with methanol and then with dichloromethane. Methanol eluates were analysed by HPLC-DAD, the dichloromethane eluates with TLC-DAD. The method was validated for precision, repeatability and accuracy. The calibration plots were linear between 0.1 and 50.0 microg/mL for all pesticides, the correlation coefficients, r, were between 0.9992 and 1.000 as determined by HPLC-DAD. In the TLC experiments, the best fit for the calibration lines was found when the calibration data were analysed using a second-degree polynomial regression. Calibration plots lay between 0.1 and 17 microg/spot for all pesticides, the correlation coefficients, r, were between 0.9974 and 0.9997 determined by TLC-DAD. The LOD was between 0.04 and 0.65 microg/spot (TLC-DAD) and between 0.02 and 3.68 microg/mL (HPLC-DAD).