TLC Separation Research Articles

Phosphatidic Acid and N-Acylphosphatidylethanolamine Form Membrane Domains in Escherichia coli Mutant Lacking Cardiolipin and Phosphatidylglycerol

The pgsA null Escherichia coli strain, UE54, lacks the major anionic phospholipids phosphatidylglycerol and cardiolipin. Despite these alterations the strain exhibits relatively normal cell division. Analysis of the UE54 phospholipids using negativeion electrospray ionization mass spectrometry resulted in identification of a new anionic phospholipid, N-acylphosphatidylethanolamine. Staining with the fluorescent dye 10-N-nonyl acridine orange revealed anionic phospholipid membrane domains at the septal and polar regions. Making UE54 null in minCDE resulted in budding off of minicells from polar domains. Analysis of lipid composition by mass spectrometry revealed that minicells relative to parent cells were significantly enriched in phosphatidic acid and N-acylphosphatidylethanolamine. Thus despite the absence of cardiolipin, which forms membrane domains at the cell pole and division sites in wild-type cells, the mutant cells still maintain polar/septal localization of anionic phospholipids. These three anionic phospholipids share common physical properties that favor polar/septal domain formation. The findings support the proposed role for anionic phospholipids in organizing amphitropic cell division proteins at specific sites on the membrane surface.

TLC Separation of Isomeric Diazinodithiins and Diazinyl Sulfides as the Smiles Rearrangement Products

The reactions of diazinodithiins 1, 5, and 9 with sodium methanethiolate in DMSO, followed by methylation with methyl iodide, proceed with or without the Smiles rearrangement to give isomeric diazinodithiins 4, 8, and 12 and diazinyl sulfides 2, 3, 6, 7, 10, and 11. The aim of this was to find TLC conditions to follow the rearrangement, to separate and identify thioazines 1–12. The retention parameter RF was measured and next calculated as separation factors ΔRF, RS, and α. The RF values were correlated with the dipole moments of thioazines 1–12 and the symmetry (C2h and C2v) of diazinodithiins 1, 4, 5, 8, 9, and 12.

TLC study of the separation of the enantiomers of lactic acid

Summary Enantiomer separation by TLC is still much less frequent than with other, mostly instrumental, chromatographic techniques. From a literature survey it is apparent that separation of the enantiomers of d,l-lactic acid is primarily of interest to the diary industry and that this particular separation is less frequently performed by chromatographic than by membrane techniques. As far as we are aware, before our studies only one report of TLC separation of the enantiomers of d,l-lactic acid was available in the literature; this is dated 1991 and describes the use of non-instrumental TLC only. In this study, we started by reproducing the TLC procedure originating from 1991, for this purpose using TLC with automatic sample application and densitometric detection. We managed to repeat the earlier procedure and to achieve full, i.e. baseline, separation of the enantiomers, with a remarkable distance between the two antimers. However, we revealed a significant drawback of this separation procedure — d-(−)-lactic acid was transported almost with the mobile-phase front and its densitometric quantification was barely possible because of the relatively high UV absorption of the mobile-phase front line. The reference method for separation of the enantiomers of d,l-lactic acid consisted in preliminary impregnation of commercial silica gel TLC plates with copper(II) acetate. In-situ formation of bidentate complexes of the d,l-lactic acid antimers with the Cu2+ cation resulted in different mobilities of these complex cations in the planar chromatographic system. The objectives of this study were twofold — to investigate separation of the enantiomers of d,l-lactic acid with other transition metal cations (i.e., Co2+, Ni2+, and Mn2+) used to impregnate the silica gel (to achieve resolution that might enable quantification of the two lactic acid antimers and not only the l-(+) enantiomer) and to gain deeper insight into the mechanism of separation with these metal cations. For purposes of comparison, we chromatographed d,l-lactic acid on non-impregnated silica gel layers. As a result, we managed to establish efficient separation conditions with the Ni2+ and Co2+ cations that outperformed the earlier established procedure involving the Cu2+ cation, and — partially at least — to elucidate the mechanism of separation of the enantiomers of d,l-lactic acid by these TLC systems. The Mn2+ cation proved unsuitable for the purpose. Finally, we managed to separate the enantiomers of d, l-lactic acid on non-impregnated silica gel layer also, which seems yet more proof of the microcrystalline chirality of silica gel used as stationary phase and of its substantial contribution to the enantiomer separation investigated.

Redundant Synthesis of Cysteinyl-tRNACys in Methanosarcina mazei

A subset of methanogenic archaea synthesize the cysteinyl-tRNA(Cys) (Cys-tRNA(Cys)) needed for protein synthesis using both a canonical cysteinyl-tRNA synthetase (CysRS) as well as a set of two enzymes that operate via a separate indirect pathway. In the indirect route, phosphoseryl-tRNA(Cys) (Sep-tRNA(Cys)) is first synthesized by phosphoseryl-tRNA synthetase (SepRS), and this misacylated intermediate is then converted to Cys-tRNA(Cys) by Sep-tRNA:Cys-tRNA synthase (SepCysS) via a pyridoxal phosphate-dependent mechanism. Here, we explore the function of all three enzymes in the mesophilic methanogen Methanosarcina mazei. The genome of M. mazei also features three distinct tRNA(Cys) isoacceptors, further indicating the unusual and complex nature of Cys-tRNA(Cys) synthesis in this organism. Comparative aminoacylation kinetics by M. mazei CysRS and SepRS reveals that each enzyme prefers a distinct tRNA(Cys) isoacceptor or pair of isoacceptors. Recognition determinants distinguishing the tRNAs are shown to reside in the globular core of the molecule. Both enzymes also require the S-adenosylmethione-dependent formation of (m1)G37 in the anticodon loop for efficient aminoacylation. We further report a new, highly sensitive assay to measure the activity of SepCysS under anaerobic conditions. With this approach, we demonstrate that SepCysS functions as a multiple-turnover catalyst with kinetic behavior similar to bacterial selenocysteine synthase and the archaeal/eukaryotic SepSecS enzyme. Together, these data suggest that both metabolic routes and all three tRNA(Cys) species in M. mazei play important roles in the cellular physiology of the organism.

Application of a new technique in two-dimensional TLC separation of multicomponent mixtures

In this publication a new technique in planar chromatography is presented. The technique connects two-dimensional chromatography with multiple gradient development. It enables separation of multicomponent mixtures such as plant extracts with simultaneous qualitative identification of components. On the same plate less polar aglycones and more polar glycoside derivatives can be identified simultaneously. The new technique was applied to analysis of anthraquinone derivatives in Rhamnus frangulae cortex methanol extract.

Crucial role of formaldehyde and its reaction products in the antiproliferative activity of some potential pesticides

A group of thirteen newly synthesized potential herbicides, N -aryltrichloroacetamides or 2-(chlorophenoxy)acyl derivatives, have been initially investigated by reversed phase (RP) TLC. The lipophilicity of the substances was described by retention factors in water, log k w , calculated from experimental RP TLC data, and by log P values calculated by use of software. Biological activity was examined by use of the BioArena system of TLC separation then (micro)biological detection. The potential role of formaldehyde (HCHO) in the toxic antibacterial action of substances against Pseudomonas savastanoi pv. phaseolicola bacterial cells was investigated. The effect of HCHO capturers (L -arginine and reduced glutathione) and Cu 2+ ions on the bioactivity and mechanism of toxicity of the substances was examined. It was established that HCHO and its potential reaction products (e.g. 1 O 2 , O 3 ) are crucial in the mechanism of action of these molecules. Correlations between the lipophilicity and bioactivity of th...

Membrane Topology and Essential Amino Acid Residues of Phs1, a 3-Hydroxyacyl-CoA Dehydratase Involved in Very Long-chain Fatty Acid Elongation*

Yeast Phs1 is the 3-hydroxyacyl-CoA dehydratase that catalyzes the third reaction of the four-step cycle in the elongation of very long-chain fatty acids (VLCFAs). In yeast, the hydrophobic backbone of sphingolipids, ceramide, consists of a long-chain base and an amide-linked C26 VLCFA. Therefore, defects in VLCFA synthesis would be expected to greatly affect sphingolipid synthesis. In fact, in this study we found that reduced Phs1 levels result in significant impairment of the conversion of ceramide to inositol phosphorylceramide. Phs1 proteins are conserved among eukaryotes, constituting a novel protein family. Phs1 family members exhibit no sequence similarity to other dehydratase families, so their active site sequence and catalytic mechanism have been completely unknown. Here, by mutating 22 residues conserved among Phs1 family members, we identified six amino acid residues important in Phs1 function, two of which (Tyr-149 and Glu-156) are indispensable. We also examined the membrane topology of Phs1 using an N-glycosylation reporter assay. Our results suggest that Phs1 is a membrane-spanning protein that traverses the membrane six times and has an N terminus and C terminus facing the cytosol. The important amino acids are concentrated in or near two of the six proposed transmembrane regions. Thus, we also propose a catalytic mechanism for Phs1 that is not unlike mechanisms used by other hydratases active in lipid synthesis.

Use of the BioArena system for indirect detection of endogenous ozone in spots after TLC or OPLC separation

Singlet oxygen ( 1 O 2 )-catalyzed oxidation of water leads to a variety of oxidants (e.g. ozone, O 3 ) in host-parasite relationships as well. It can be detected indirectly in TLC or OPLC zones by use of the simple BioArena system and O 3 -eliminating molecules (e.g. d -limonene and indigo carmine) in the culture medium. It follows from these new findings that not only formaldehyde but also O 3 and related bioreactive compounds may play a crucial role in the mechanism of antibacterial activity of antibiotic-like compounds. The toxic potential of a molecule, however, originates from the ratio of the oxidants produced in the chromatographic spots.

4-(D-Glucosamino)-7-nitrobenzoxadiazole: synthesis, anomers, spectra, TLC behavior, and applications

A new synthesis of the known fluorescent compound 3 (4-(D-glucosamino)-7-nitro-2,1,3- benzoxadiazol-4-yl) is reported, starting from D-glucosamine and non-fluorescent 4-aryloxy-7- nitrobenzofurazans, 4a-e, 5. The α and β anomers are easily interconverted but can be separated by TLC (Rf β > Rf α). The non-fluorescent new congener 8 {2-(N-(2',4',6'-trinitrophenyl)- amino)-2-deoxy-D-glucose} and the related known compound 9 {2-(N-(2',4'-dinitrophenyl)- amino)-2-deoxy-D-glucose} have anomers that may be seen in NMR spectra, but are too rapidly interconverted for TLC separation. The UV-Vis and fluorescence spectra of 3 depend markedly on solvent polarity. The TLC method allows the analytical determination of glucosamine from pharmaceutical preparations by conversion into 3 and detection by its fluorescence.

Matching IR-MALDI-o-TOF Mass Spectrometry with the TLC Overlay Binding Assay and Its Clinical Application for Tracing Tumor-Associated Glycosphingolipids in Hepatocellular and Pancreatic Cancer

Glycosphingolipids (GSLs), composed of a hydrophilic carbohydrate chain and a lipophilic ceramide anchor, play pivotal roles in countless biological processes, including the development of cancer. As part of the investigation of the vertebrate glycome, GSL analysis is undergoing rapid expansion owing to the application of modern mass spectrometry. Here we introduce direct coupling of IR-MALDI-o-TOF mass spectrometry with the TLC overlay binding assay for the structural characterization of GSLs. We matched three complementary methods including (i) TLC separation of GSLs, (ii) their detection with oligosaccharide-specific proteins, and (iii) in situ MS analysis of protein-detected GSLs. The high specificity and sensitivity is demonstrated by use of antibodies, bacterial toxins, and a plant lectin. The procedure works on a nanogram scale, and detection limits of less than 1 ng at its best of immunostained GSLs were obtained. Furthermore, only crude lipid extracts of biological sources are required for TLC-IR-MALDI-MS, omitting any laborious GSL downstream purification procedures. This strategy was successfully applied to the identification of cancer-associated GSLs in human hepatocellular and pancreatic tumors. Thus, the in situ TLC-IR-MALDI-MS of immunolabeled GSLs opens new doors by delivering specific structural information of trace quantities of GSLs with only a limited investment in sample preparation.

Herbal remedies for the management of seed-borne fungal pathogens by an edible plant Decalepis hamiltonii (Wight & Arn)

Antifungal activity-guided assay of solvent extracts of Decalepis hamiltonii (Wight & Arn) (Asclepiadaceae) against important phytopathogenic fungi, known to cause diseases in sorghum, maize and paddy proved to be highly significant. Among the five solvent extracts tested, Petroleum ether extract showed highly significant antifungal activity. Phytochemical analysis revealed that the antifungal active principle is a phenolic compound. TLC separation of the phenolic fraction using chloroform as an eluting solvent revealed the presence of seven bands but the antifungal activity was observed only in band five with Rf value 0.77. The antifungal active compound is identified as 2-hydroxy-4-methoxybenzaldehyde based on Nuclear Magnetic Resonance (NMR) and mass spectral analysis. The Minimal inhibitory concentration (MIC) varied between 200 μg ml−1 and 700 μg ml−1 depending on the fungal species. Seed treatment of the active principle significantly increased seed germination and seed vigour with a corresponding decrease in seed mycoflora. The antifungal active compound was effective against all the 24 fungal species tested suggesting broad-spectrum antifungal activity. Comparative evaluation of the active principle with the synthetic fungicides revealed that the antifungal activity of the active principle obtained from the plant is better than that of synthetic fungicide. This plant being an edible one can be exploited in the management of seed-borne pathogenic fungi and the prevention of biodeterioration of grains and mycotoxin elaboration during storage.

Optimization of the TLC separation of seven amino acids

A ternary mobile phase for thin-layer chromatographic separation and identification of seven amino acids on microcrystalline cellulose, with ninhydrin as detection reagent, has been optimized by use of the experimental design software packages Design-Expert 6 and Statistica . When the results from each product were compared the optimum mobile phase was found to be butanol-glacial acetic acid-water 60.14:18.77:21.09. The performance of this mobile phase was confirmed experimentally.

Supercritical fluid extraction for quality control in beer industry

The knowledge of lipid composition in beer ingredients (malt and corn grits) and wort enables the quality control for final product. Since supercritical fluid extraction (SFE) is an efficient technique for preparing samples for analysis without the use of solvents, in this research Supercritical CO 2 (SC–CO 2) extraction was compared with the traditional Soxhlet one for a gravimetric determination of total lipids on malt and corn grits. The obtained extracts were then analyzed by HPLC-ELSD after TLC separation of triacylglycerols (TAGs) for lipids fingerprint. The extraction of total fats achieved by a 60-min run with pure CO 2 at 65 MPa and 100 °C was 43% higher than that produced by Soxhlet performed for 9 h for malt. The extraction was intermediate for SFE at 60 and 80 °C. The recovery of the TAG obtained with SC–CO 2 at 100 °C was statistically comparable with results from Soxhlet extraction.

Isolation and characterization of a novel uronic acid-containing acidic glycosphingolipid from the ascidian Halocynthia roretzi

A novel uronic acid-containing glycosphingolipid (UGL-1) was isolated from the ascidian Halocynthia roretzi. UGL-1 was prepared from chloroform-methanol extracts and purified by the use of successive column chromatography on DEAE-Sephadex, Florisil, and Iatrobeads. Chemical structural analysis was performed using methylation analysis, gas chromatography, gas chromatography-mass spectrometry, matrix-assisted laser-desorption/ionization time-of-flight mass spectrometry, and 1H-NMR spectra. The chemical structure of UGL-1 was determined to be a glucuronic acid-containing glycosphingolipid, Galbeta1-4(Fucalpha1-3)GlcAbeta1-1Cer. The ceramide component was composed of C16:0 and C18:0 acids and C16-, C17-, and C18-phytosphingosines as major components.

Development and Validation of Spectrophotometric, TLC and HPLC Methods for the Determination of Lamotrigine in Presence of Its Impurity

Three reliable, rapid and selective methods have been developed and validated for the determination of lamotrigine in the presence of its impurity, 2,3-dichlorobenzoic acid. The first method is spectrophotometric method using p-chloranilic acid forming a colored product with lambda(max) 519+/-2 nm. All variables affecting the reaction have been investigated and the conditions were optimized. Beer's law was obeyed over a concentration range of 10-200 microg ml(-1) with mean accuracy 100.13+/-0.44%. The molar ratio of the formed ion-association complex is found to be 1 : 1 as deduced by Job's method. The conditional stability constant (K(f)), standard free energy (DeltaG), molar absorptivity(epsilon), and sensitivity index were evaluated. The second method is based on TLC separation of the cited drug (Rf=0.75+/-0.01) from its impurity (Rf=0.23+/-0.01) followed by densitometric measurement of the intact drug spots at 275 nm. The separation was carried on silica gel plates using ethyl acetate : methanol : ammonia 35% (17 : 2 : 1 v/v/v) as a mobile phase. The linearity range was 0.5-10 microg/spot with mean accuracy 99.99+/-1.33%. The third method is accurate and sensitive stability-indicating HPLC method based on separation of lamotrigine from its impurity on a reversed phase C(18) column, using a mobile phase of acetonitrile : methanol : 0.01 M potassium orthophosphate (pH 6.7+/-0.1) (30 : 20 : 50 v/v/v) at ambient temperature 25+/-5 degrees C and UV detection at 275 nm in an overall analysis time of about 6 min., based on peak area. The injection repeatability, intraday and interday repeatability were calculated. The procedure provided a linear response over the concentration range 1-12 microg ml(-1) with mean accuracy of 99.50+/-1.30%. The proposed methods were successfully applied for the determination of lamotrigine in bulk powder, in dosage form and in presence of its impurity. The results obtained were analyzed by ANOVA to assess that no significant difference between each of the three methods and the reported one. The validation was performed according to USP guidelines.

A Bifunctional Δ12,Δ15-Desaturase from Acanthamoeba castellanii Directs the Synthesis of Highly Unusual n-1 Series Unsaturated Fatty Acids

The free-living soil protozoon Acanthamoeba castellanii synthesizes a range of polyunsaturated fatty acids, the balance of which can be altered by environmental changes. We have isolated and functionally characterized in yeast a microsomal desaturase from A. castellanii, which catalyzes the sequential conversion of C(16) and C(18) Delta9-monounsaturated fatty acids to di- and tri-unsaturated forms. In the case of C(16) substrates, this bifunctional A. castellanii Delta12,Delta15-desaturase generated a highly unusual fatty acid, hexadecatrienoic acid (16:3Delta(9,12,15)(n-1)). The identification of a desaturase, which can catalyze the insertion of a double bond between the terminal two carbons of a fatty acid represents a new addition to desaturase functionality and plasticity. We have also co-expressed in yeast the A. castellanii bifunctional Delta12,Delta15-desaturase with a microsomal Delta6-desaturase, resulting in the synthesis of the highly unsaturated C(16) fatty acid hexadecatetraenoic acid (16:4Delta(6,9,12,15)(n-1)), previously only reported in marine microorganisms. Our work therefore demonstrates the feasibility of the heterologous synthesis of polyunsaturated fatty acids of the n-1 series. The presence of a bifunctional Delta12,Delta15-desaturase in A. castellanii is also considered with reference to the evolution of desaturases and the lineage of this protist.

Determination of Veterinary Pharmaceuticals in Production Wastewater by HPTLC-Videodensitometry

An SPE-HPTLC method for simultaneous identification and quantification of seven pharmaceuticals in production wastewater was optimized and validated. The studied compounds were enrofloxacine, oxytetracycline, trimethoprim, sulfamethazine, sulfadiazine, sulfaguanidine and penicillin G/procaine. The method involves solid-phase extraction on hydrophilic-lipophilic balance cartridges with methanol and HPTLC analysis of extracts on CN modified chromatographic plates followed by videodensitometry at 254 and 366 nm. Optimization of chromatographic separation was performed by systematic variation of the mobile phase composition using genetic algorithm approach and the optimum mobile phase composition for TLC separation was 0.05 M H2C2O4:methanol = 0.81:0.19 (v/v). Linearity of the method was demonstrated in the ranges from 1.5 to 15.0 µg L−1 for enrofloxacine, 100–500 µg L−1 for oxytetracycline, 150–600 µg L−1 for trimethoprim, 300–1100 µg L−1 for sulfaguanidine and 100–400 µg L−1 for sulfamethazine, sulfadiazine and penicillin G/procaine with coefficients of determination higher than 0.991. Mean recoveries ranged from 74.6 to 117.1% and 55.1 to 108.0% for wellspring water and production wastewater, respectively. Only sulfaguanidine showed lower results. The described method has been applied to the determination of pharmaceuticals in wastewater samples from pharmaceutical industry.

Determination of methionine in pharmaceuticals after chromatographic separation

A simple and accurate analytical procedure is proposed for determination of methionine after chromatographic separation. Optimum conditions were established for TLC separation of methionine from other biologically active compounds. Spectrophotometric quantification was based on oxidation of leuco xylene cyanol FF to its blue form, xylene cyanol FF. The absorbance of the xylene cyanol FF dye formed was measured at λ = 613 nm. The procedure was used for determination of methionine in complex pharmaceutical preparations.

Oxylipin Profiling of the Hypersensitive Response in Arabidopsis thaliana: FORMATION OF A NOVEL OXO-PHYTODIENOIC ACID-CONTAINING GALACTOLIPID, ARABIDOPSIDE E

Oxylipin Profiling of the Hypersensitive Response in Arabidopsis thaliana: FORMATION OF A NOVEL OXO-PHYTODIENOIC ACID-CONTAINING GALACTOLIPID, ARABIDOPSIDE E

Smooth Muscle Relaxant Triterpenoid Glycosides from Rubus idaeus (Raspberry) Leaves

A methanol extract of Rubus idaeus leaves had a relaxant activity on transmurally stimulated Guinea pig ileum preparations in vitro. The extract, when fractionated by column chromatography, yielded two distinct bands with pharmacological activity. After extensive column and preparative TLC separation, several compounds with smooth muscle relaxant properties were obtained. From band two, three triterpenoid glycosides were characterized, which had significant muscle relaxant activity. From spectroscopic and spectrometric data, these were identified as 3β-(O-β-D-glucopyranosyl)-olean-12-ene-1α, 2α, 3β-triol (TG1); 28-(O-β-D-glucopyranosyl)-urs-12-ene-2α, 3β, 19α-trihydroxy-28-oic acid (TG2); and 3β-(O-β-D-glucopyranosyl)-olean-12-ene-1α, 2α, 3β-trihydroxy-28-oic acid (TG3). To the best of our knowledge, TG1 and TG3 are novel compounds.