Showing papers on "Thin-layer chromatography published in 2010"

Isolation, purity, analysis and stability of hyperforin as a standard material from Hypericum perforatum L

Abstract: In 1996 131.5 million daily doses of preparations containing extracts of Hypericum perforatum L. were prescribed in Germany for treating mild to moderately severe depressive disorders. New pharmacological and clinical results focus on hyperforin as the main active ingredient of the drug. Hyperforin (C35H52O4) is one of the main components (2-4%) of the dried herb Hypericum perforatum L. It was isolated after six consecutive steps: extraction of deep-frozen blossoms (-20 degrees C) with n-hexane by means of an Ultra Turrax at room temperature; separation of lipophilic substances on a silica gel column; purification of the relevant fraction by preparative HPLC; evaporation of the mobile phase under reduced pressure; removal of the remaining water by freeze-drying; and storage of hyperforin at -20 degrees C under nitrogen. The identity and purity of the isolated substance were determined by high-performance thin-layer chromatography (HPTLC), high-performance liquid chromatography (HPLC) with diode-array and ultraviolet detection (DAD and UV), Fourier-transformed infrared (FTIR) and proton nuclear magnetic resonance (1H NMR) spectroscopy, and liquid chromatography coupled with positive-ion electrospray-ionization tandem mass spectrometry (LC-ESI(+)-MS-MS). By use of these methods the purity of hyperforin was shown to be >99.9%. Peroxides present at each step of the isolation were detected by titration and by means of Merckoquant analytical peroxide test-strips. Elimination of the peroxides and stabilization of hyperforin was achieved by consistent protection from oxidation-the mobile phases were protected by use of ascorbic acid; evaporation and freeze-drying were performed under nitrogen; and the mobile phase used for preparative HPLC was sparged with helium. Stability testing was performed by HPLC-the samples were stored at -30 degrees C in a normal atmosphere and at -20, 4, and 20 degrees C in a normal atmosphere or under nitrogen. Results were compared with those obtained after storage under liquid nitrogen (-196 degrees C). Because of its high sensitivity to oxidation, hyperforin was more stable under nitrogen under all test conditions. There was no statistically significant difference between results obtained after 8 months at -20 degrees C under nitrogen or at -30 degrees C under a normal atmosphere and those from the reference sample stored under liquid nitrogen (-196 degrees C). Despite this, because of the tendency of hyperforin to degrade, long-term storage at -70 degrees C under nitrogen is recommended.

Marine Bacterium Derived Lipopeptides: Characterization and Cytotoxic Activity Against Cancer Cell Lines

Abstract: A marine Bacillus circulans DMS-2 was able to grow and produce biosurfactant on glucose mineral salts medium (GMSM) with a reduction in the surface tension up to 27 mN m−1. The microorganism produced 1.64 ± 0.1 g l−1 of crude biosurfactant. The lipopeptide nature of the produced biosurfactant was confirmed by primulin and ninhydrin assays using High Performance Thin Layer Chromatography (HPTLC). Preparative thin layer chromatography (TLC) was performed to purify the lipopeptides from the crude biosurfactant. The critical micelle concentrations (CMC) of the crude and purified products were found to be 90 and 40 mg l−1 respectively. Fourier transform infrared spectrophotometer (FTIR) and matrix assisted laser desorption/ionization time of flight (MALDI-ToF) mass spectral analysis revealed the identity of the produced lipopeptides as surfactin (m/z 1,023 Da) and fengycin (m/z 1,495 Da) isoforms. The purified marine lipopeptides displayed a significant antiproliferative activity against the human colon cancer cell lines HCT-15 (IC50 80 μg ml−1) and HT-29 (IC50 120 μg ml−1).

Thin-Layer Chromatographic Analysis of Carotenoids in Plant and Animal Samples

Abstract: Summary Carotenoids are among the most widespread and important pigments in living organisms. They are found in common foods and vegetables. The characteristic pattern of alternating single and double bonds in the polyene backbone enables them to absorb excess energy from other molecules. The nature of the specific end groups on carotenoids may effect their polarity, thus solubility ranges from acetone to hexane. Because of this wide range of polarity, specific extraction and separation procedures are required. In these procedures use of planar chromatography in food analysis might seem a minor aspect of carotenoid analysis. This review describes available data on analysis of carotenoids by thin-layer chromatography (TLC). It has been found that petroleum ether, acetone, and hexane are the major mobile phases used for TLC. Thin-layer chromatography was found to have the potential to be the first choice for analysis of carotenoids in biological samples. The uses of other, orthogonal chromatographic methods, for example HPLC, spectroscopy (mass spectroscopy), scanning densitometry, and image analysis with TLC can enable precise analysis of carotenoids.

Pre-staining thin layer chromatography method for amino acid detection

Abstract: Thin layer chromatography (TLC) is an analytical method that is used commonly. It usually includes five steps: spotting, separating, drying, spraying/immersing and color development. In this paper, TLC was modified with pre-staining method, which only consisted of 3 steps: spotting, separating and color development. The modified thin layer chromatography can be used for the analysis of amino acids. When compared to the classical thin layer chromatography, the improved method was more rapid and inexpensive and the results obtained were clean and reproducible. However, it is suitable for the high throughput screening of amino acid-producing strains. Key words: Thin layer chromatography, pre-staining, amino acid detection.

Thin-layer chromatography combined with MALDI-TOF-MS and 31P-NMR to study possible selective bindings of phospholipids to silica gel.

Abstract: High-performance thin-layer chromatography (HPTLC) is a highly established separation method in the field of lipid and (particularly) phospholipid (PL) research. HPTLC is not only used to identify certain lipids in a mixture but also to isolate lipids (preparative TLC). To do this, the lipids are separated and subsequently re-eluted from the silica gel. Unfortunately, it is not yet known whether all PLs are eluted to the same extent or whether some lipids bind selectively to the silica gel. It is also not known whether differences in the fatty acyl compositions affect the affinities to the stationary phase. We have tried to clarify these questions by using a readily available extract from hen egg yolk as a selected example of a lipid mixture. After separation, the complete lanes or selected spots were eluted from the silica gel and investigated by a combination of MALDI-TOF MS and 31P NMR spectroscopy. The data obtained were compared with the composition of the total extract (without HPTLC). Although there were significant, solvent-dependent losses in the amount of each lipid, the relative composition of the mixture remained constant; there were also only very slight changes in the fatty acyl compositions of the individual PL classes. Therefore, lipid isolation by TLC may be used without any risk of major sample alterations.

Normal silica gel and reversed phase thin-layer chromatography coupled with UV spectroscopy and IR-MALDI-o-TOF-MS for the detection of tetracycline antibiotics.

Abstract: Tetracyclines (TCs) form a group of bacteriostatic antibiotics with closely related structures and similar chemical and physicochemical properties. They are widely employed as therapeutics in human and veterinary medicine. Here, we introduce the combination of UV spectroscopic detection of high-performance thin-layer chromatography (HPTLC)-separated TCs with direct analysis on solid phase using infrared matrix-assisted laser desorption/ionization orthogonal time-of-flight mass spectrometry (IR-MALDI-o-TOF-MS). Normal silica gel phase- and water-wettable hybrid C18 reversed phase layers (RP-18W) both allowed HPTLC separation and sensitive UV spectroscopic detection followed by MS analysis of distinct TCs bands using the liquid matrix glycerol. The novel approach of direct IR-MALDI-o-TOF-MS analysis resulted in the unequivocal identification of four structurally different TCs employed in this study, and linear calibration curves were produced for analyte amounts from 20 ng to 1 μg. MS analysis of TCs from RP-18W HPTLC plates was found to be superior when compared to the spectra acquired from the silica gel layer. Ionic analytes obtained from the RP-18W surface are mainly detected as protonated species of high abundance accompanied by a reduced formation of adducts as well as background ions arising from the matrix or the stationary phase. This results in decreased complexity of the spectra and enhanced sensitivity of the combinatorial method. An approximate detection limit of 5 ng of individual TCs in mixtures by combining RP-18W HPTLC with IR-MALDI-o-TOF-MS offers a novel timely and cost-efficient method for tracing TCs.

Using one-dimensional (1D) and two-dimensional (2D) quantitative proton (1H) nuclear magnetic resonance spectroscopy (q NMR) for the identification and quantification of taste compounds in raw onion (Allium cepa L.) bulbs and in aqueous solutions where onion tissues are soaked.

Abstract: Solutions obtained by soaking onion (Allium cepa L.) bulbs samples in water are frequently consumed, either directly or as part of dishes, both at home or in the food industry. However, little information is available regarding the extracted metabolites and the extraction mechanisms. In this article, the composition of raw onion extracts and of aqueous solutions where raw onion tissues were soaked was investigated directly by quantitative proton nuclear magnetic resonance spectroscopy (q (1)H NMR). The assignment of NMR signals was performed, with less than 3% (in area) of unidentified peaks. Analyses of one-dimensional (1)H NMR spectra with additional two-dimensional NMR studies showed 20 regions of interest where 3 saccharides, 17 amino acids, and 5 organic acids were detected and quantified. Resonance assignment with chemical shift was done for each saccharide, as well as for each amino acid and organic acid, with additional work on spin-spin coupling pattern and on observed and not observed correlations from correlation spectroscopy studies. Quantification of saccharides was performed and qualified by works on peak decomposition algorithms. Complementary studies by high-performance liquid chromatography, mass spectroscopy and tandem mass spectroscopy, and thin layer chromatography and preparative layer chromatography were carried out in order to validate the NMR results on identification.

Direct analysis of reversed-phase high-performance thin layer chromatography separated tryptic protein digests using a liquid microjunction surface sampling probe/electrospray ionization mass spectrometry system.

Abstract: The sampling, ionization and detection of tryptic peptides separated in one-dimension on reversed-phase high-performance thin layer chromatography (HPTLC) plates was performed using liquid microjunction surface sampling probe electrospray ionization mass spectrometry. Tryptic digests of five proteins [cytochrome c, myoglobin, beta-casein, lysozyme and bovine serum albumin (BSA)] were spotted on reversed phase HPTLC RP-8 F254s and HPTLC RP-18 F254s plates. The plates were then developed using 70/30 methanol/water with 0.1M ammonium acetate. A dual purpose extraction/electrospray solution containing 70/30/0.1 water/methanol/formic acid was infused through the sampling probe during analysis of the developed lanes. Both full scan mass spectra and data dependent tandem mass spectra were acquired for each development lane to detect and verify the peptide distributions. Data dependent tandem mass spectra provided both protein identification and sequence coverage information. Highest sequence coverages were achieved for cytochrome c and myoglobin (62.5% and 58.3%, respectively) on reversed phase RP-8 plates. While the tryptic peptides were separated enough for identification, the peptide bands did show some overlap with most peptides located in the lower half of the development lane. Proteins whose peptides were more separated gave higher sequence coverage. Larger proteins such as beta-casein and BSA which were spotted in lower relative amounts gave much lower sequence coverage than the smaller proteins.

Resolution of multicomponent mixture of amino acids using environmentally benign eluents: A green chromatographic approach

Abstract: A new green TLC has been used for identifying and monitoring the migration behavior of amino acids through silica and kieselguhr static flat bed in contact of n-butyl alcohol, ethyl acetate or ethylene glycol and their mixtures. From the point of view of chromatographic performance, a mixture of n-butyl alcohol-70% aqueous ethylene glycol-ethyl acetate ratio 5:3:2 by volume proves to be more efficient than the individual components for separation of amino acids from their binary, ternary and quaternary mixtures and the chromatographic parameters like ΔR(F) , separation factor (α) and resolution (R(S) ) for the separation were calculated. Effect of the presence of foreign substances such as metal cations, anions, vitamins and pesticides as impurities in the sample on the separation was also examined. Effect of substitution of butanol by various alcohols has been examined to assess the impact of hydrophobicity of alcohols on the separation of amino acids. The limits of detection for tyrosine, tryptophan, alanine, isoleucine, methionine and serine were found to be 0.10 μg/spot, whereas for lysine it is 0.05 μg/spot. Application of the selected TLC system for the identification and separation of amino acids present in drugs/pharmaceuticals has been performed.

TLC separation of amino acids with a green mobile phase

Abstract: Thin-layer chromatography (TLC) of fifteen amino acids was performed using silica gel and alumina impregnated with micellar solutions of cetrimide and cetylpyridinium chloride as stationary phases and aqueous solutions of dextrose as mobile phases. TLC system comprising of silica gel impregnated with micellar solution of cetrimide (5.0 mM) as stationary phase and 40% ( w/v ) aqueous solution of dextrose as mobile phase was found the most favourable for the separation of amino acids. Impregnation of silica gel with the micellar solution of cetrimide brings about a substantial change in the mobility of lysine. Separation of lysine (ketogenic) from arginine (glucogenic) is important physiologically. Surface modification of silica gel on impregnation, as indicated by FTIR and SEM studies, was responsible for improved chromatographic performance. The effect on the separation of the presence in the sample of heavy metal cations, as impurities, was examined. Limits of detection for lysine and arginine were 0.17 ...

Separation of homologous cerebrosides by silica gel column and thin‐layer chromatography

Abstract: Cerebroside mixtures isolated from beef spinal cord lipids can be chromato-graphically resolved into twelve or more components. The technique employed was multiple thin-layer chromatography with a newly developed solvent. A fractionation of the cerebrosides could also be obtained by silicic acid column chromatography using chloroform-methanol mixtures. The fractions were characterized (a) by thin-layer chromatography; (b) by gas chromatography of the fatty acid methyl esters obtained after methanolysis of the cerebrosides. It could be shown that the chromatographic behaviour of cerebrosides is decisively influenced by the nature of their fatty acid component.

Isolation and Identification of the Amines Resulting From Chemical and Catalytic Reduction of 20‐Hydroxyimino‐Steroids (Restatement)

Abstract: Sodium‐propanol reduction of 3β‐hydroxy‐5‐pregnen‐20‐one oxime affords the two 20‐amino epimers in a ratio 20 α to 20 β equal to 1.3, while hydrogenation with platinum in acetic acid gives a mixture of the two corresponding saturated amines in the ratio of 2:1. Quantitative separation of 20‐amino epimers is conveniently achieved by chromatography on silica gel. Thin layer chromatography, optical rotatory dispersion, infrared absorption, nuclear magnetic resonance data are joined. Copyright © 1967 Wiley‐VCH Verlag GmbH & Co. KGaA, Weinheim

Evaluation of Antioxidant Activities and Active Compounds Separated from Water Soluble Extracts of Korean Black Pine Barks

Abstract: Black pine barks from the sout hern region of Korea were extracted using pressurized hot water and the water soluble extracts were then separated in a stepwise fashion using a variety of solvents, column chromatography (CC), thin layer chromatography (TLC), and high pressure liquid chromatography (HPLC). The antioxidant activities of each fraction and the active compounds were determined based on the radical scavenging activities of 2,2-diphenyl-1-picrylhydrazyl (DPPH), reductive potential of ferric ion, and total phenol contents. A DPPH test showed that the half maximal effective concentration (EC

Thin layer chromatography plates and related methods

Abstract: In an embodiment, a method for manufacturing a thin layer chromatography (“TLC”) plate is disclosed. The method includes forming a layer of elongated nanostructures (e.g., carbon nanotubes), and at least partially coating the elongated nanostructures with a coating. The coating includes a stationary phase and/or precursor of a stationary phase for use in chromatography. The stationary phase may be functionalized with hydroxyl groups by exposure to acidified water vapor or immersion in a concentrated acid bath (e.g., HCl and methanol). At least a portion of the elongated nanostructures may be removed after being coated. Embodiments for TLC plates and related methods are also disclosed.

Iodine speciation using thin-layer chromatography coupled to inductively coupled plasma-mass spectrometry by means of an extraction device

Abstract: Thin-layer chromatography (TLC) has been coupled to inductively coupled plasma-mass spectrometry (ICP-MS) using an extraction device, which allows a nearly complete recovery of compounds from thin-layer chromatograms within short extraction times, small volumes and without contamination. Iodinated X-ray contrast agents were separated and analyzed with the newly developed setup. Iodine speciation analysis was carried out with deviations between the expected and the measured concentrations in spiked urine samples from 3.2 to 16.6%.

Separation, purification, and characterization of analogues components of a commercial sample of new Fuchsin.

Abstract: New Fuchsin (NF), also known as Magenta III, has potential applications in photodynamic therapy. The commercial product labeled NF contains two other dye components in different proportions, Magenta II and Magenta I (Rosaniline). The proportions of NF, Magenta II, and Magenta I determined by reversed-phase high-performance liquid chromatography (RP-HPLC) in the commercial sample used were 71.6 +/- 0.4%, 25.2 +/- 0.2%, and 2.8 +/- 0.1% (n = 7), respectively. The isolation, purification, and characterization of commercial NF dye components were carried out applying different techniques, such as preparative column liquid chromatography (PCLC), thin layer chromatography (TLC), RP-HPLC, absorption spectrophotometry, nuclear magnetic resonance spectroscopy (NMR), electrospray ionization mass spectrometry (ESI-MS), and tandem electrospray ionization mass spectrometry (ESI-MS-MS). After separation and isolation, the degree of purity obtained for NF compound was higher than 95% and 92% for Magenta II and Magenta I compounds, respectively. Therefore, it is essential to ensure a high degree of purity of these dyes as raw material to obtain new drugs intended for therapeutic treatments.

Theoretical Basis of Thin Layer Chromatography (TLC)

Abstract: In column chromatography a defined sample amount is injected into a flowing mobile phase. The mix of sample and mobile phase then migrates through the column. If the separation conditions are arranged such that the migration rate of the sample components is different then a separation is obtained. Often a target compound (analyte) has to be separated from all other compounds present in the sample, in which case it is merely sufficient to choose conditions where the analyte migration rate is different from all other compounds. In a properly selected system, all the compounds will leave the column one after the other and then move through the detector. Their signals, therefore, are registered in sequential order as a chromatogram. Column chromatographic methods always work in sequence. When the sample is injected, chromatographic separation occurs and is measured. This type of chromatography is known as “Online Chromatography”.

Quantitative determination of phenolic acids in lonicera japonica thunb. using high performance thin layer chromatography

Abstract: A new, simple, precise, and rapid high performance thin layer chromatography (HPTLC) method has been developed and validated for the simultaneous quantitative determination of three phenolic acids, namely, 3,5-O-dicaffeoylquinic acid, 3,4-O-dicaffeoylquinic acid and chlorogenic acid from flower buds of Lonicera japonica. The separation of these compounds was carried out on silica gel 60F254 eluted with ethyl acetate:chloroform:methanol:formic acid:water (6:2.5:1.5:0.6:0.1 v/v/v/v) and a detection wavelength at 330 nm. The developed method gave a good linear regression relationship between peak area and an observed concentration range of 200–1200 ng spot−1 for the three aforementioned marker compounds. Spike recoveries were within 97.81–101.59% and the RSD values of precision were in the range of 0.65–3.21%. The method presented good results for the following parameters: selectivity, linearity, accuracy, precision, robustness, as well as limit of detection and limit of quantification. Therefore, this metho...

Separation of unsaturated dinitrophenylhydrazones into classes

Abstract: Unsaturated dinitrophenylhydrazones with one or more isolated double bonds may, as a class, be separated from saturated, α-β-unsaturated or α-β, γ-δ-unsaturated DNPHs by using silicagel impregnated with AgNO3 (column chromatography) or alumina impregnated with AgNO3 (thin layer chromatography).

Chemical Constituents from Rhizomes of Curculigo breviscapa

Abstract: UV-2401PC spectrometer was used to obtain the UV spectra in methanol (MeOH). IR spectra were recorded on Nexus 870-FT-IR spectrophotometer with potassium bromide (KBr) pellets. Mass spectrometry (MS) was performed on VG Autospec-3000 spectrometer and API- QSTAR-Pulsar-1 spectrometer using a positive ion model. 1D and 2D NMR spectra were measured on Bruker AM-400 spec-trometer with TMS as an internal standard. Column chromato-graphy was carried out on Sephadex LH-20 gel (25 - 100 µm, Pharmacia Fine Chemical Co. Ltd.). Thin layer chromatography (TLC) was carried out on silica gel G precoated plates (Qingdao Haiyang Chemical Co. Ltd.) and spots were detected by spray-ing with 5% H

Partial Purification of Antimicrobial Compounds Isolated from Mycelia of Tropical Lentinus cladopus LC4

Abstract: Lentinus cladopus LC4 produced at least eight antimicrobial compounds (ACs) which are active against plant and human pathogens. Three ACs in its crude mycelial were extracted with methanol and partial purification was carried out with silicic acid column chromatography and by thin layer chromatography (PTLC). The antimicrobial activity was tested by paper disc method and antibiographic method. The chromatography purification eluted with dichloromethane containing 5% methanol gave one active fraction (FII). This fraction which was active against X. campestris pv. glycines and showing two inhibition zones against Bacillus subtilis on bioautographic plates with the R f s 0.8 and 0.7. FI and FIII fractions eluted with dichloromethane containing 0 and 10% methanol performed one inhibition zone with R f s 0.8 and 0.7 respectively. However, their activities were lower than that of FII fraction. The PLTC purification gave one separate fraction with R f value of 0.73 and it was active against X. campestris pv. glycines. The compound of R f 0.73 fraction should be further studied using TLC and HPLC to obtain the pure substance for molecule characterization.

Chromatography-bioluminescence coupling reveals surprising bioactivity of inthomycin A

Abstract: By employing a novel technique for the direct coupling of a bacterial bioassay with chromatography, we discovered a gyrA promotor active compound in myxobacterial extracts and elucidated the structure directly without any isolation step. As a result, we identified inthomycin A as the bioactive substance. Our method is based on a whole-cell bioluminescent reporter gene assay coupled with thin-layer chromatography for primary hit detection and with liquid chromatography (LC)/mass spectrometry or LC/NMR for dereplication and structure elucidation. Previously, inthomycin A has been isolated from Streptomycetes and was associated with the inhibition of cellulose biosynthesis and herbicidal activity. Thus, our findings support the basic principle that completely different microbial phyla are able to synthesize the same natural product. Moreover, our results indicate that inthomycin A affects bacterial DNA supercoiling, which reveals an unexpected bioactivity for this compound. These results can possibly promote further investigation of the biosynthesis as well as the biological activity of inthomycins and related natural products.