Thin-layer Chromatography Sheets Research Articles

Visible fluorescent sensing of Cu2+ ions in urine by reusable chitosan/l-histidine–stabilized silicon nanoparticles integrated thin layer chromatography sheet

This study reports a facile approach for the fabrication of chitosan (CS, biopolymer)– and l-histidine (L-His, biomolecule)–stabilized self-assembled silicon nanoparticles (SiNPs) for sensing Cu2+ ions. Approached method yielded 3.8 ± 0.04 nm size CS/L-His–SiNPs particles, with high stability against harsh pH and temperature conditions. Besides, CS/L-His–SiNPs highly selective to Copper amongst different metal ions tested (Fe3+, Mg2+, Al3+, Cr3+, Cr6+, Cu2+, Mn2+, Cd2+, Pb2+, Zn2+, Hg2+, Ca2+, Li2+, Po42−, As3+, As5+). As compared to the blank-SiNPs (LOD = 96.49 ± 0.223 μM) and CS-SiNPs (LOD = 33.35 ± 1.004 μM); L-His ligand, enhanced the sensitivity of the CS/L-His–SiNPs toward Cu2+ with remarkable LOD value of 55.02 ± 0.42 nM. Applicability of CS/L-His–SiNPs was evaluated by coating CS/L-His–SiNPs on thin layer chromatography (TLC) sheets, CS/L-His–SiNPs–TLC sheets exhibited significant sensing capacity toward Cu2+ ions, with a detection range of 4.0–900 μM, making them suitable for on-site analysis of Cu2+ ions from both environmental and clinical samples. Finally, Cu2+ sensing practicality of CS/L-His–SiNPs–TLC sheets were challenged against real human urine samples. Expressively, CS/L-His–SiNPs–TLC sheets could be regenerated using ethylenediaminetetraacetic acid (EDTA), without losing their photostability, and can be reused further.

TLC-MALDI-TOF-MS-based identification of flavonoid compounds using an inorganic matrix.

Thin layer chromatography (TLC) is frequently used to obtain the fingerprint of a plant extract. Although the retardation factor and the response to visualisation give primary information about compound identification, the direct TLC-mass spectrometry (MS) coupling allows a more detailed characterisation of samples. To demonstrate the potential for the flavonoid dereplication using an inorganic matrix-assisted laser desorption ionisation time-of-flight mass spectrometry (MALDI-TOF-MS) method with and without TLC separation. Samples derived from wine, apple or rose were deposited on an aluminium-backed silica gel TLC sheet compatible with the MS adapter. Unlike the wine sample, for apple and rose samples compound derivatisation was necessary. These two samples were deposited twice and the plate was cut in two parts. One half was oversprayed with Neu-Peg reagent to visualise flavonoids while the inorganic matrix was deposited on each flavonoid zone on the second half for MS ionisation. Mass spectra obtained for samples without plate development showed numerous ions corresponding to glycosylated flavonoids. The lower m/z observed could be due either to aglycone flavonoids or to in-source fragment ions. After plate development, a separation of many spots was observed and each spot was analysed separately leading to a deeper identification of the present flavonoids. Moreover, isobaric flavonoids with different hRf values could be differentiated. TLC-MALDI-TOF-MS using an inorganic matrix enabled the analysis of anthocyanins in positive mode and of flavonols, flavanols, dihydrochalcones and phenolic acids in negative mode, reducing adduct, aggregate forms giving thus simple and reliable spectra for the dereplication approach of flavonoids in complex samples.

EVALUATION OF α-GLUCOSIDASE INHIBITORY POTENTIAL OF METHANOLIC LEAF EXTRACT OF OCIMUM CANUM

Objective: The present investigation was designed to study the inhibitory effects of methanolic leaf extract of Ocimum canum (O. canum) on α-glucosidase using in vitro model followed by an assessment of bioactive compounds.Methods: The methanolic leaf extract was prepared by Soxhlet extraction method and partially purified by thin layer chromatography (TLC). Each band was subjected to α-glucosidase inhibition study. The positive bands were further characterized by high-performance liquid chromatography (HPLC) and quadrupole time of flight (Q-TOF) micro mass spectrometer.Results: Out of the several combinations of solvent systems, toluene, ethyl acetate and formic acid combination in the ratio of 7:2:1 revealed 5 bands on the TLC sheet. Among all the TLC bands, 2 bands (band A and B) showed the significant inhibitory effect on α-glucosidase activity. HPLC analysis of band A and B revealed the presence of two important polyphenolic compounds, namely rosmarinic acid (RA) and ursolic acid (UA). Q-TOF micromass spectrometer analysis revealed the percentage availability of RA, caffeic acid, tartaric acid, quercetin and other polyphenolic components in the bioactive bands.Conclusion: The study revealed that methanolic leaf extract of O. canum exhibits potent inhibition of α-glucosidase activity. Inhibition of α-glucosidase activity might be attributed to the presence of the polyphenolic compounds like RA and UA. Therefore, this finding can lead to the development of natural α-glucosidase inhibitors by the O. canum leaf extract.

Review of Advances in Planar Radiochromatography

The materials, techniques, and instruments for the detection and quantification of radiolabeled compounds by planar radiochromatography are described. Included is sample preparation; separations on Instant Thin Layer Chromatography sheets, thin layer plates, and chromatography papers; and detection and quantification by film autoradiography, liquid scintillation counting, storage phosphor imaging, and in situ radioactivity scanning. These methods are widely applied in radiopharmaceutical purity determination, metabolism studies, and many other types of chemical, biochemical, and biological investigations.

Thin-layer chromatography combined with diode laser thermal vaporization inductively coupled plasma mass spectrometry

Here we present a novel coupling of thin-layer chromatography (TLC) to diode laser thermal vaporization inductively coupled plasma mass spectrometry (DLTV ICP MS). DLTV is a new technique of aerosol generation which uses a diode laser to induce pyrolysis of a substrate. In this case the cellulose stationary phase on aluminum-backed TLC sheets overprinted with black ink to absorb laser light. The experimental arrangement relies on economic instrumentation: an 808-nm 1.2-W continuous-wave infrared diode laser attached to a syringe pump serving as the movable stage. Using a glass tubular cell, the entire length of a TLC separation channel is scanned. The 8-cm long lanes were scanned in ∼35s. The TLC – DLTV ICP MS coupling is demonstrated on the separation of four cobalamins (hydroxo-; adenosyl-; cyano-; and methylcobalamin) with limits of detection ∼2pg and repeatability ∼15% for each individual species.

クロマトグラフィー学習のための携帯用実験教材の試作と実践

A portable kit for performing thin layer chromatography (TLC) was prepared as an experimental tool for learning chromatography in and outside of class. This kit mainly consisted of the following components : (1) two plastic eye drop bottles containing water and ethanol, respectively (for developing or extracting solvents) ; (2) aluminum TLC sheets (15×35mm) precoated with octadecylsilanized silica gel ; (3) a plastic push top vial for development chamber ; (4) a screw top glass vial containing colored bath powder for the extraction of dyes. The better test scores of the students using the experimental kit on the review test for chromatography suggested the kit was effective for better understanding of chromatographic behavior.

Therapeutic Drug Monitoring of Nevirapine in Resource‐Limited Settings

We developed a simple and inexpensive thin-layer chromatography (TLC) assay for semiquantitative detection of saliva concentrations of nevirapine in resource-limited settings. The method was validated in an African target population. Paired plasma and saliva nevirapine concentrations were assayed by high-performance liquid chromatography (HPLC); saliva concentrations of nevirapine were also assayed by TLC. The rate of false-positive results was the proportion of subtherapeutic nevirapine saliva and plasma concentrations determined by HPLC that were judged to be therapeutic in saliva specimens by TLC. The rate of false-negative results was the proportion of therapeutic nevirapine saliva and plasma concentrations determined by HPLC that were judged to be subtherapeutic in saliva specimens by TLC. The extent of agreement in TLC readings between 5 technicians and 2 batches of TLC sheets was evaluated. Twenty-five (9%) of 286 African adults had a subtherapeutic plasma nevirapine concentration. The median ratio of nevirapine concentrations in saliva to those in plasma was 0.51:1. The rate of false-positive results for TLC was 0% (0 of 23 specimens) when TLC results were compared with HPLC results for saliva specimens and 8% (2 of 25 specimens) when TLC results were compared with HPLC results for plasma specimens. The rate of false-negative results for TLC was 1% (3 of 263 specimens) when TLC results were compared with HPLC results for saliva specimens and 1% (3 of 261 specimens) when TLC results were compared with HPLC results for plasma specimens. The extent of agreement of TLC results was substantial for the 5 technicians (Fleiss's kappa = 0.77) and for the 2 batches of sheets (Cohen's kappa = 0.80). The TLC assay was found to be sensitive, specific, and robust in the detection of subtherapeutic nevirapine concentrations in saliva specimens obtained from African HIV-infected adults. It is an attractive alternative to HPLC for therapeutic drug monitoring of nevirapine in resource-limited settings.

Sex pheromones in three Bradysia species (Dipt., Sciaridae): novel bioassays with female body extracts and fractions

AbstractFemales of the fungus gnat species Bradysia difformis, Bradysia optata and Bradysia tilicola are able to produce sexual attractants. In males, the female extracts elicit species‐specific courtship behaviour and attraction in an olfactometer bioassay. Attractiveness of the sex pheromone extracts seems to differ in the investigated species, being highly attractive for males in B. difformis and only moderately attractive in B. tilicola. Female B. difformis are attractive from late pupal stages until death of the imago. In contrast, female B. optata and B. tilicola are not attractive to males before emergence from the pupa and attractiveness decreases in older females. The pheromones seem to be located on the surface of several body compartments of freshly emerged females. Sexual pheromones of all three species represent multicomponent blends and can be separated in several fractions via thin layer chromatography (TLC). Males of all species show courtship behaviour when placed on developed TLC sheets, indicating the position of the biologically active compounds. In B. difformis and B. optata, the most non‐polar TLC fraction is attractive to males, whereas the single fractions of B. tilicola‐pheromone elicited almost no attraction of males.

Separation and identification of multicomponent mixture by thin-layer chromatography coupled with Fourier transform–infrared microscopy

A fast and convenient method based on coupled thin-layer chromatography (TLC) with Fourier transform infrared (FTIR) microscopy has been established for separation and identification of multicomponent mixtures. In this study, the method was developed and consummated with more perfect TLC spots transferral process and consistent FTIR testing conditions. A newly developed technique, solid-phase extract (SPE) was introduced for sample pre-treatment instead of using traditional column chromatography. It is a new field for SPE that has already been widely applied in many other fields. It not only overcomes the backwards (low separation efficiency, time consuming and solvent consumption) of column chromatography but also makes it much easier to choose an optimum TLC sheet and to set suitable TLC loading. With all the above-mentioned modifications and supplements, the analytical method of coupled TLC with FTIR microscopy for separation and identification of multicomponent mixtures becomes more convenient and more efficient. In addition, a very complex sample (a die-cast release agent) was used as an example to demonstrate the technique.

Application of pressure sensitive paint measurement to a low-solidity cascade diffuser of a transonic centrifugal compressor

A pressure sensitive paint (PSP) measurement has been known as a pressure field measurement technique based on the oxygen quenching phenomenon of luminescence of specific luminophores. A PSP measurement was applied for pressure field measurement in a low-solidity circular cascade diffuser of a single-stage transonic centrifugal compressor with 5 in pressure ratio for HFC134a gas. The oxygen concentration was about 500 ppm. Ru (bath-phen) was adsorbed on a silica-gel thin-layer chromatography sheet, and the sheet was pasted onto the side-wall between the cascade vanes. A drastic change in luminescent intensity was recognized during a surge condition. Also the pressure variations based on luminescent intensity agreed well with the pressure fluctuations measured using a semiconductor pressure sensor with high-frequency-response. It was shown that a PSP measurement worked well to investigate the unsteady pressure fields in a circular cascade diffuser of a transonic centrifugal compressor. Moreover, the time response of PSP becomes clear as a problem to be overcome for the present.

感圧塗料を用いた遷音速遠心圧縮機内圧力場の可視化

A pressure sensitive paint (PSP) measurement is recently known as a pressure field measurement technique based on oxygen quenching phenomenon of luminescence of specific luminophores. A PSP measurement was applied to a pressure field measurement in a low-solidity circular cascade diffuser of a single-stage transonic centrifugal compressor at 17, 800 rpm with 5 in pressure ratio for HFC134a gas. The oxygen concentration was about 500 ppm based on the measured velocity of sound of the gas. Ru (bath-phen) was used as a PSP. The PSP was adsorbed on a silica-gel thin-layer chromatography sheet, and the sheet was fixed on the side-wall between the cascade vanes. The luminescent intensity drastically changed during a surge condition. And the pressure variations based on luminescent intensity agreed well with the pressure fluctuations measured using semiconductor pressure sensors with high-frequencyresponse at three points. It was shown that a PSP measurement worked well to investigate unsteady pressure fields in a transonic centrifugal compressor. However, there are still some problems to be overcome.

On the coupling of fluorescence line-narrowing spectroscopy and poly(ethylene)imine-cellulose thin-layer chromatography

The feasibility of a direct coupling of the low-temperature, high-resolution, fluorescence line-narrowing spectroscopy (FLNS) technique and the autoradiographic 32 P -post-labelling technique for DNA adducts is explored. FLNS has been used to identify analytes on various types of thin-layer chromatography (TLC) plates, but not on poly(ethylene)imine-cellulose (PEI-cellulose) TLC sheets involved in the 32 P -post-labelling technique which includes a two-dimensional separation. PEI-cellulose was found to exhibit a high fluorescence background. Its fluorescence excitation and emission characteristics were studied in detail and the signal-to-noise ratios of the FLNS spectra were optimized using background subtraction, time-resolved detection and a new synchronous scanning procedure. Benzo[a]pyrene (B[a]P) tetrols and a standard B[a]P–DNA adduct were used as model compounds. Limits of detection were in the low-picomole range. The technique was applied for the identification of spots extracted from PEI-cellulose TLC sheets obtained after a 32 P -post-labelling procedure of DNA containing B[a]P adducts at a level of two adducts per hundred bases.

Semiautomated Application of Steroids onto Thin-Layer Chromatography Sheets

Semiautomated Application of Steroids onto Thin-Layer Chromatography Sheets

Development of a radiometric spot-wash assay for squalene synthase

The principle of selective elution from a solid phase has been exploited to develop an assay for the determination of squalene biosynthesis in rat liver homogenates. Using either [1- 14C]isopentenyl diphosphate as a precursor for squalene or [2- 14C]farnesyl diphosphate as a direct substrate of squalene synthase, the production of radiolabeled squalene is determined after adsorption of assay mixtures onto silica gel thin-layer chromatography sheets and selective elution of the diphosphate precursors into a solution of sodium dodecyl sulfate at alkaline pH. The use of [2- 14C]farnesyl diphosphate, and of an endogenous oxygen consumption system (ascorbate/ascorbate oxidase) to prevent further metabolism of squalene, allows the method to be applied as a dedicated assay for squalene synthase activity. The assay has been developed in microtiter plate format and may be deployed either in a quantitative, low-throughput mode or in a qualitative, high-throughput mode. The latter is suitable for screening to aid in the discovery of new inhibitors of squalene synthase.

Separation of peptides on Empore thin-layer chromatography sheets and blotting onto polyvinylidene difluoride membranes with subsequent gas phase sequencing.

Methods for the separation of peptides on a new type of thin-layer chromatography (TLC) sheet and blotting onto polyvinylidene difluoride (PVDF) membranes with subsequent gas phase sequencing are described. For validation, the A and B chain of insulin were chromatographed on Empore TLC sheets and either extracted or blotted onto PVDF membranes. The advantages and disadvantages of thin-layer chromatography on Empore sheets versus high performance liquid chromatography (HPLC) are discussed, along with the possibility of combining the two methods. In addition, TLC was combined with electrophoresis (fingerprinting) for the separation of complex peptide mixtures. Blotting from TLC sheets onto PVDF membranes was performed in two ways: contact diffusion and electrophoretic transfer. In our experiments electroblotting was more effective. Amino acid sequence determination of the B chain of insulin was possible both after extraction from the TLC sheet and after blotting onto PVDF membranes. In the former case, liquid phase sequencing and, in the latter case, gas phase sequencing was performed. The possibility to blot from TLC sheets onto membranes, e.g. PVDF, may prove useful in many fields, for example in biochemistry, and in molecular and cell biology.

Direct use of Empore sheets in overpressured thin-layer chromatography

The impregnation of the edges of plates before carrying out forced-flow thin-layer chromatography (TLC) as overpressured TLC is time consuming. This paper reports the optimisation of analytical parameters and the direct use of a new separation medium, Empore TLC sheets, for forced-flow TLC. The pressure of the water pump ( P) and the flow-rate of the eluent ( F) were studied and optimum values of P = 2–4 bar and F = 0.4–0.6 ml/min were determined. The separation of a mixture of dyes and a bergamot oil was investigated and the results demonstrated that the direct utilisation of Empore TLC sheets in forced-flow TLC gave performances similar to pre-impregnated high-performance TLC plates.

Metabolism of xylitol in dental plaque.

It has been reported previously that xylitol added to glucose used to challenge dental plaque in vivo caused a reduced acid formation. The aim of the present study was to approach the mechanism by which xylitol may affect glucose catabolism in plaque bacteria. Suspensions of freshly collected 4-day-old plaque bacteria were incubated, one batch with labeled xylitol, one with labeled glucose, in vitro at 37 degrees C. Samples of cells were taken out at time intervals, collected on paper discs and subjected to scintillation counting. It was observed that the plaque bacteria took up xylitol, the uptake increasing with incubation of more than 3-4 h, whereas the same cells took up glucose immediately. Cells which had taken up xylitol were extracted with boiling water, extracts concentrated and applied on thin-layer chromatography sheets. A radioactive component with mobility like xylitol-5-phosphate was isolated from the cell extracts, and also a component where labeled xylitol was associated with macromolecules. It is suggested that the accumulation of the metabolities within the cells inhibits glycolysis.

Direct cell culture assay of thin-layer-chromatographed material without prior substance elution

A method enabling the direct assay in a cell culture system of material separated by thinlayer chromatography (TLC) without prior elution of active material from the TLC sheet is described. After chromatographic development on a plastic cellulose sheet, small disks were punched out from the sheet and transferred to microwells used for cell culture. Lymphoid cells in culture medium were incubated with the disks and the DNA synthesis was measured by [ 3H]thymidine incorporation. The reliability of the method was tested with l-alanine and thymidine, which are known to enhance the DNA synthesis of lymphoid cells and to interfere with the uptake of labeled thymidine, respectively. The incorporation of [ 3H]thymidine was stimulated with disks containing l-alanine and inhibited with disks containing unlabeled thymidine, completely in agreement with the expected results. Elution experiments showed that more than 75% of l-alanine and thymidine on the TLC disks was eluted into the culture medium within 1 h. Elution of material of higher molecular weight was also demonstrated. The method was used for the separation and assay of a thymocyte-specific growth factor isolated from calf thymus and greatly facilitated the detection of the active material after TLC.

Topochemistry of trace metals in nasal mucosa Potentialities of some histochemical methods and energy dispersive X-ray microanalysis

Histochemical methods and energy dispersive X-ray micro-analysis (EDX-analysis) were evaluated in model experiments and on tissue sections for their usefulness in detecting traces of metals in biological tissue. The goal for this study was to establish a method for localization of nickel deposits in the nasal mucosa, where it has been found in concentrations between 1 and 40 microgram/g in nickel exposed individuals. The histochemical methods tested were staining with dimethylglyoxime, rubeanic acid and dithizone, the Turnbull and Prussian blue methods and TIMM'S sulphide silver procedure. In model experiments nickel-, cobalt-, copper-, zinc- and ironsalts were applied to thin-layer chromatography sheets (TLC-sheets) and stained by the histochemical methods. Spots containing 500 and 50 ng of these metals represented the smallest amounts that could consistently be detected in these experiments, except for the sulphide silver method which seemed a little more sensitive. With the latter method, moreover, zinc was detected in 40 micrometer thick cryostat sections of gelatine made up with 1 microgram/g of the metal. For nickel the corresponding figure was 10 to 50 microgram/g. On specimens of nasal mucosa from nickel-exposed workers, a faint colour was obtained in 40 micron thick cryostat sections from specimens that had been immersed in dithizone, but the colour was too weak for histological analysis. None of the other coloured chelating agents caused noticeable staining when applied to blocks or to cryostat sections. TIMM'S sulphide silver method caused strong staining of the basal layers of the surface epithelium and of fibroblast-like cells in the underlying connective tissue. This staining pattern is described in more detail in a separate report. Rat liver tissue was analyzed by atomic absorption before and after araldite embedding. Blocks of gelatine made up with nickel, copper, zinc and iron were embedded in epoxy resin and analyzed by atomic absorption. Large changes in the metal concentrations, usually an increase, were found after embedding. Ultrathin sections from this material were used to test the sensitivity of the EDX-equipment. Referring to the concentrations determined by atomic absorption in the embedded material, iron was detected at 1215 microgram/g and 362 microgram/g (gelatine standards) but not at 167 microgram/g (rat liver). Similar values could not be determined for nickel, copper or zinc, because of background radiation resulting from the presence of these metals in the instrument. We did not succeed in establishing a procedure for detecting nickel deposits in nasal mucosa with any of the methods which were tested. The most sensitive but least specific of the tested methods for visualizing heavy metals in the nasal mucosa, was TIMM'S sulphide silver procedure. The preparation of tissue for this method is discussed.

Micro assay for 3-hdyroxy-3-methylglutaryl-CoA reductase in rat liver and in L-cell fibroblasts

A rapid, highly reproducible, micro-incubation, direct thin-layer chromatographic assay for 3-hydroxy-3-methylglutaryl-CoA reductase is described. The isolation and quantitation of mevalonic acid is simplified by direct application of the deproteinized, acidified incubation mixture to thin-layer chromatography sheets. The tedious and variable extraction of mevalonic acid into ether is eliminated by this procedure. Background levels are lower than those obtained with comparable assays and permit quantitation of as little as 30 pmoles of mevalonic acid. The method is illustrated by analysis of 3-hydroxy-3-methylglutaryl CoA reductase from rat liver, cultured L cells, and solubilized microsomal reductase. The rapid inactivation of reductase by MgATP and the low levels of reductase activity in cholesterol-treated L cells are accurately determined by using the micro-incubation, direct thin-layer chromatography method.