Development Thin-layer Chromatography Research Articles

Industrial wastewater analysis: a toxicity-directed approach

Methods of toxicity-directed analysis have been developed for the characterization and identification of toxic organic constituents in industrial wastewater. Sequential solid-phase extraction is followed by high-performance liquid chromatography (HPLC) fractionation or by automated multiple development thin-layer chromatography fractionation (AMD–TLC) of the toxic extracts. Toxic fractions were finally analyzed by gas chromatography–mass spectroscopy (GC–MS). Toxicity was detected before each of the analytical steps by the bioluminescence inhibition of Vibrio fischeri, which was performed on microtiter plates and on the developed TLC plates. While sequential extraction broadens the polarity range of the procedure, the new variants of the luminescence test make the method very versatile and fast. The potential of this kind of toxicity-directed analysis with respect to resolution and polarity of analytes is discussed and applications to partial effluents of a tannery, to molasses wastewater and a spent dyeing bath are presented. A variety of benzothiazoles and more polar organics were identified as major toxic compounds in tannery effluents. It is shown that the procedures are well suited to detect individual toxic components in complex industrial wastewaters. The use of LC-MS is proposed to extend the polarity range of the final identification step. ©

Automated multiple development thin layer chromatography of some plant extracts

The separation of ten plant extracts using automated multiple development thin-layer chromatography (AMD-TLC) is described. Alcoholic extracts were obtained from Cinchona succirubra, Aesculus hippocastanum, Berberis vulgaris, Artemisia abrotanum, Carduus marianus, Thuja occidentalis, Baptisia tinctoria, Paulinia cupana, Lycopus europaeus and Echinacea angustifolia. The separation was performed on silica plates (Sil G-50 UV 254 (Macherey—Nagel), 10 × 20 cm). AMD was achieved in 25 steps using methanol, ethyl acetate, toluene, 1,2-dichloroethane, 25% ammonia solution and anhydrous formic acid as modifiers. The chromatograms were evaluated with a Shimadzu CS-9000 dual-wavelength flying-spot scanner. Better separations were obtained using AMD than isocratic elution.

Computeraided optimization of gradient multiple development thin-layer chromatography III. Multi-stage development over a constant distance

A theoretical model of multiple development over a constant distance is presented. The model is verified with experimental results. The final RF values are calculated by computer program for known retention vs. eluent composition relationships for the solutes. The predicted and experimental RF values were compared and showed satisfactory agreement.

TLC Developing Chamber for Screening Column Chromatography Fractions

Barring the use of very expensive automatic monitoring equipment, the successful separation of colorless substances requires some means of screening the eluate fractions so that those of like composition may be combined. This process is customarily accomplished by thin-layer chromatography (TLC). We describe here a novel TLC development chamber that greatly enhances the efficiency of this task.

Analysis of plant extracts by multiple development thin-layer chromatography

Abstract The simplest version of multiple gradient development, two-stage development, is discussed. A computer program for calculation of final R F values in this gradient mode for known retention vs . eluent composition relationships for different plant extracts was used. Comparison of predicted and experimental R F values showed satisfactory agreement.

Computer-aided optimization of gradient multiple development thin-layer chromatography: Part II. Multi-stage development

A theoretical model of gradient multiple development is presented as a basis for the optimization of separation by planar multi-step development and au

Demonstration of Membrane Association and Surface Location of Mycoplasma pneumoniae Antigens Using Monoclonal Antibodies

We examined 10 monoclonal antibodies (mAbs) directed against Mycoplasma pneumoniae proteins of 200, 170, 67, 46 and 42 kDa, and one mAb directed against a glycolipid component. The membrane association of the antigens reacting with our mAbs was investigated, in particular by phase-fractionation involving use of the detergent Triton X-114. The 170 kDa protein was shown to be membrane-associated, and surface exposure of this antigen was demonstrated by its disappearance from SDS-PAGE patterns after treatment of intact mycoplasmas with proteolytic enzymes. Cross-reactions with protein antigens of Mycoplasma genitalium were also shown. A mAb directed against a component of a lipid extract, prepared by the method used for preparation of the antigen used in the complement fixation (CF) test for serological diagnosis of M. pneumoniae infection, reacted with one major and a few minor bands in thin-layer chromatography (TLC) of the crude extract. The glycolipid character of this major antigen was demonstrated by treatment of the extract with sodium periodate, and by development of the TLC with orcinol/ferric chloride. These reactive bands were the same as those detected by the use of polyclonal mouse antiserum and a human convalescent serum, a result showing that the CF antigen contains a glycolipid moiety reacting with our mAb. The surface exposure of this antigen was demonstrated by binding of mAbs to intact cells.

Stepwise gradient development in thin-layer chromatography : IV. Miniaturized generators for continuous and stepwise gradients

A miniaturized generator of continuous gradients for equilibrium sandwich chambers with a glass distributor is described. The gradient profile is controlled by the initial concentration of the weaker eluent and its initial volume and by the composition and rate of delivery of the stronger eluent. Stepwise gradients are generated by delivery of eluents of increasing concentration of the stronger solvent from reservoirs in accordance with the gradient programme. Comparison of densitograms of thin-layer chromatograms from isocratic and gradient elution (continuous or stepwise) showed the considerable improvement in separation under gradient elution conditions.

Short bed-continuous development thin-layer chromatography of glycosphingolipids.

The technique of short bed--continuous development chromatography has been utilized to increase the separation of glycosphingolipids on high performance thin-layer chromatography plates. The theoretical goal of increasing separation of bands by decreasing solvent strength was achieved within a practical time span as a result of the high solvent velocities in the short bed tank. Examples are given for increased separation of short and long chain neutral glycolipids, acetylated neutral glycolipids, and gangliosides.

Stepwise gradient development in thin-layer chromatography : I. Optimization of gradient program

The application of an equilibrium sandwich chamber with a glass distributer for stepwise gradient development in thin-layer chromatography is described. Six to ten fractions of the eluent having increasing concentrations of a polar modifier are programmed graphically and introduced directly under the distributer with a micropipette. A comparison of the gradient profile with the obtained chromatogram permits adjustment of the gradient program in order to improve the positions of the spots on the chromatogram. The technique is relatively simple and permits the use of multicomponent gradients; it is especially suitable for the separation of complex samples, e.g., plant extracts, including zonal micropreparative separation. It is possible to improve the spot separation in a fragment of the plate without changing the positions of the remaining well separated spots.

Inexpensive instrument for programmed multiple development in thin-layer chromatography

A programmed multiple thin-layer chromatography (TLC) developer of new design has been constructed and tested. It is controlled by a programmable delay timer and supported by other common solid-state components. The system is dedicated to ‘‘Mode 1’’ operation and features a hard-wired drying interval. Solvent removal is effected by a cold-air blower and the elution chamber includes a solvent pump. This ensures rapid solvent vapor saturation of the TLC atmosphere after a drying cycle. The instrument is relatively inexpensive and can be constructed with facilities provided by many laboratories.

Stepwise gradient development in thin-layer chromatography : III. A computer program for the simulation of stepwise gradient elution

A general equation for the final R F value of a solute chromatographed under conditions of stepwise gradient elution with one void volume of mobile phase has been derived. The elution process and the distance travelled by the spot as a function of eluent volume are illustrated graphically for retention—eluent composition relationships typical of a displacement adsorption mechanism or for reversed-phase chromatography. A computer program (in BASIC) is given for the simulation of stepwise gradient thin-layer chromatography. The program can be used for the optimization of stepwise gradient programs by computer simulation of the elution process.

Improved high-performance liquid chromatography assay of doxorubicin: detection of circulating aglycones in human plasma and comparison with thin-layer chromatography.

We compared doxorubicin and metabolite pharmacokinetic data obtained from thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC) assay of plasma samples from six patients who had been treated with doxorubicin. Duplicate 1-ml samples were extracted with chloroform: isopropanol (1:1) and assayed using a sensitive HPLC system incorporating a dual pump gradient with tetrahydrofuran as the mobile phase and fluorescence detection. Duplicate 1-ml samples from the same specimens were assayed using a modification of a previously described TLC assay. Areas under the curve for doxorubicin by HPLC (3.36 +/- 2.30 microM X h) and TLC (4.16 +/- 2.50 microM X h) were not significantly different (P = 0.5). Terminal half-life of doxorubicin by HPLC (28.0 +/- 6.98 h) and TLC (23.2 +/- 7.8) (P = 0.29) and the calculated total-body clearances by HPLC (0.55 +/- 0.29 l/min) and TLC (0.45 +/- 0.23) (P = 0.55) were not significantly different. Areas under the curve for doxorubicinol by HPLC (2.75 +/- 1.4 microM X h) and TLC (2.53 +/- 7.1 microM X h) (P = 0.73) showed no significant differences. HPLC detected a mixed 7-deoxydoxorubicinol aglycone-doxorubicin aglycone peak, 7-deoxydoxorubicin aglycone, and two nonpolar, unidentified metabolites. TLC detected the following aglycone metabolites: doxorubicin aglycone, doxorubicinol aglycone, 7-deoxydoxorubicinol aglycone, an unidentified polar metabolite, and several unidentified nonpolar metabolites. From these data we conclude that HPLC and TLC detect concentrations of doxorubicin and doxorubicinol from human plasma equally well to concentrations of 7.0 nM (4 pmol injected doxorubicin). Aglycones do circulate in human plasma at concentrations above the detection limits of both assays. Doxorubicinol aglycone, which is detected by TLC but not by HPLC, may be formed from artifactual breakdown of doxorubicinol during TLC development. Unidentified nonpolar compounds seen on HPLC and TLC may represent further doxorubicin metabolism than previously described.

Contemporary developments in thin-layer chromatography

Qualitative and quantitative applications of thin-layer chromatography (TLC) in the biomedical and pharmaceutical fields are briefly surveyed. Some recent developments, including high-performance TLC (HPTLC) and reversed-phase TLC (RPTLC), together with techniques such as multiple development and continuous development, are considered. Instrumentation for HPTLC, and new developments in the detection and spectroscopic characterization of substances on TLC plates, are briefly described.

Correlation in continuous development thin-layer chromatography

Various relationships in continuous development TLC using a binary solvent system are discussed. These include the dependanc of analysis time and required plate length on the shape of a ZRfvs. solvent composition curve and the dependendancy of analysis time on the spot separation required.

Overlapping resolution maps as an aid in parallel development thin-layer chromatography

A method is described for constructing overlapping resolution maps for thin-layer chromatography. The axes of the maps are time, length and binary solvent composition. These maps can be used to identify sets of compounds within a mixture such that each set is fully separated under appropriate conditions. The separation of a mixture of fifteen steroids is described, using this technique.

Separation of complex mixtures by parallel development thin-layer chromatography

Description d'une methode de separation de melanges complexes par developpements paralleles simultanes, resultant en un temps relativement court de separation. Les methodes de calcul utilisees pour la determination des conditions optimales pour des developpements paralleles peuvent etre etendues a la prevision des conditions optimales pour la chromatographie en couche mince bidimensionnelle

Determination of pentoxifylline and its major metabolites in microbial extracts by thin-layer and high-performance liquid chromatography

Thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC) methods have been developed for the determination of the xanthine drug, pentoxifylline, and three of its metabolites (a secondary alcohol and two carboxylic acids) in microbial extracts. The methods require initial extraction of acidified media with dichloromethane—2-propanol (4:1). Extracts are submitted to TLC development on silica gel G layers using three solvents and HPLC development on an C 18 column using methano—phosphoric acid (0.02 M, pH 5) (3:7) as mobile phase. All systems provide good separations of the drug and its metabolites. Quantitative analyses of pentoxifylline and its metabolites by HPLC were accurate and precise. The HPLC method was applied to studies of the metabolism of pentoxyfylline by two microorganisms.

Recent Developments in Thin-Layer Chromatography - II

Abstract This review updates the one we did 4 years ago. The emphasis is on technique rather than application. Recent advances in adsorbents, plate shape, developing chambers, gradient elution and quantification methods are reviewed and commented on. Theoretical studies of the basic aspects of TLC processes are also reviewed.

Specific Programmed Multiple Development–Thin Layer Chromatography of Furazolidone in Chicken, Turkey, Swine, and Bovine Tissues: Collaborative Study

Abstract A specific method for assay of furazolidone at 2 ppb has been developed using programmed multiple development-thin layer chromatography (PMD-TLC) and the conversion of the drug to a fluorescent species which is quantitated by fluorodensitometry on the TLC plate. The method requires only 5 g tissue, specifies an automatic spotter for PMD-TLC, and is capable of measuring <1 ng of drug when the fluorodensitometer is coupled with a computing integrator. This procedure requires an average of 2 man-hours per sample and can be completed in one day. Six laboratories collaboratively studied the method for assay of furazolidone at the 2, 3, and 6 ppb levels in chicken, turkey, swine, and bovine tissues. Tissues were fortified by each laboratory and then processed through extraction, solvent partition, programmed multiple development-thin layer chromatography, and fluorodensitometry. Results showed satisfactory recoveries and accuracy. A statistical comparison of the results demonstrated that 5 of the 6 laboratories obtained similar results. The higher responses from the sixth laboratory appeared to be due to an exceptional vacuum system and the use of silated flasks. The mean drug recovery response for the tissues spiked at 2 ppb from the 5 laboratories was 2.2324 ng ± 20.2% (SD), which at the 99.7% confidence level gives no overlap with control tissue data. The results can be expected to be repeatable within and among laboratories.