Showing papers on "Thin-layer chromatography published in 2004"

Methods for analysis of conjugated linoleic acids and trans-18:1 isomers in dairy fats by using a combination of gas chromatography, silver-ion thin-layer chromatography/gas chromatography, and silver-ion liquid chromatography.

Abstract: Conjugated linoleic acids (CLA) are octadecadienoic acids (18:2) that have a conjugated double-bond system. Interest in these compounds has expanded since CLA were found to be associated with a number of physiological and pathological responses such as cancer, metastases, atherosclerosis, diabetes, immunity, and body fat/protein composition. The main sources of these conjugated fatty acids are dairy fats. Rumen bacteria convert polyunsaturated fatty acids, especially linoleic and linolenic acids, to CLA and numerous trans- containing mono- and diunsaturated fatty acids. It has been established that an additional route of CLA synthesis in ruminants and monogastric animals, including humans, occurs via delta9 desaturation of the trans-18:1 isomers. To date, a total of 6 positional CLA isomers have been found in dairy fats, each occurring in 4 geometric forms (cis,trans; trans,cis; cis,cis; and trans,trans) for a total of 24. All of these CLA isomers can be resolved only by a combination of gas chromatography (GC), using 100 m highly polar capillary columns, and silver-ion liquid chromatography, using 3 of these 25 cm columns in series. Complete analysis of all the trans-18:1 isomers requires prior isolation of trans monoenes by silver-ion thin-layer chromatography (TLC), followed by GC analysis using the same 100 m capillary columns operated at low temperatures starting from 120 degrees C. These analytical techniques are required to assess the purity of commercial CLA preparations, because their purity will affect the interpretation of any physiological and/or biochemical response obtained. Prior assessment of CLA preparations by TLC is also recommended to determine the presence of any other impurities. The availability of pure CLA isomers will permit the evaluation and analysis of individual CLA isomers for their nutritional and biological activity in model systems, animals, and humans. These techniques are also essential to evaluate dairy fats for their content of specific CLA isomers and to help design experimental diets to increase the level of the desired CLA isomers in dairy fats. These improved techniques are further required to evaluate the CLA profile in monogastric animals fed commercial CLA preparations for CLA enrichment of animal products. This is particularly important because absorption and metabolism will alter the ingested-CLA profile in the animal fed.

Analysis of Low Molecular Weight Carbohydrates in Food and Beverages: A Review

Abstract: This review presents a study of new advances in chromatographic methods and capillary electrophoresis for the analysis and quantification of carbohydrates in food and drink that have been made in essentially the last seven years (1995–2002). All the main groups of sugars have been considered: monosaccharides, disaccharides, trisaccharides, oligosaccharides, related compounds such as alditols, alditol glycosides, polyols, amino sugars, deoxy sugars, uronic acids and aldonic acids. The chromatographic methods referred to are High Performance Liquid Chromatography (HPLC), Gas Chromatography (GC) and related techniques (AMD-HPTLC: High Performance Thin-Layer Chromatography with Automated Multiple Development). Capillary Electrophoresis (CE) was also discussed.

Overview of methods for the determination of trans fatty acids by gas chromatography, silver-ion thin-layer chromatography, silver-ion liquid chromatography, and gas chromatography/mass spectrometry.

Abstract: trans Isomers of naturally occurring cis-unsaturated fatty acids are produced when liquid vegetable oils or marine oils are partially hydrogenated to produce margarine, shortenings, and other hardened-fat products. Isomeric trans fatty acids are also formed in the intestinal tract of ruminants, and they appear in small amounts in dairy products and ruminant meat. Currently, satisfactory analyses for the fatty acid profiles of fats containing trans fatty acids are obtained by gas chromatography (GC) using capillary columns coated with highly polar cyanosilicone stationary phases. In capillary GC methods, the key limitation has been the incomplete separation of trans-monoenoic acid isomers from their cis isomers; however, recent reports have demonstrated that improvements in separation are attainable with the use of 100 m columns. In these columns, there is very little overlap of cis and trans isomers. More accurate trans fatty acid analyses can be obtained by coupling GC with either silver-nitrate thin-layer chromatography or silver-nitrate liquid chromatography.

An Improved Method for the Extraction and Thin-Layer Chromatography of Chlorophyll a and b from Spinach

Abstract: A direct method of extracting plant pigments from spinach leaves into a dry organic solvent is presented. A powder is obtained by grinding a mixture of raw spinach, drying agent, and sand (1:1:2) that can be extracted directly into acetone in 10 minutes to provide a sample suitable for chromatographic analysis. This method avoids liquid–liquid extractions and subsequent drying as is found in previously reported methods. Removal of the magnesium ion from chlorophyll pigments was observed by treating the acetone extract with a strong ion-exchange resin for three minutes. The loss of magnesium ion is often observed as a degradation product of chlorophyll samples. An improved solvent system consisting of petroleum ether, cyclohexane, ethyl acetate, acetone, and methanol in 60%, 16%, 10%, 10%, and 4% quantities, respectively, is also reported. TLC separation using this extraction method and solvent system allows for the clear separation of chlorophyll a and b from all other extracted pigments.

Fast screening of low molecular weight compounds by thin-layer chromatography and "on-spot" MALDI-TOF mass spectrometry.

Abstract: Fast screening of low-MW compounds is performed by thin-layer chromatography (TLC) followed by direct on-spot matrix-assisted laser desorption/ionization time-of-flight mass spectrometry identification with nearly “matrix-free” mass spectra using an UV-absorbing ionic liquid matrix. Owing to minimal background ions from the proton donor triethylamine/α-cyano-4-hydroxycinnamic acid ionic liquid matrix, three arborescidine alkaloids, the anesthesics levobupivacaine and mepivacaine, and the antibiotic tetracycline were readily characterized most frequently by the MS detection of their protonated molecules. The technique is fast and sensitive, requires little sample preparation and manipulation, and is therefore suitable for fast screening with TLC separation and MS identification of low-MW compounds, with potential applications in areas such as phytochemistry, synthetic chemistry, and product manufacturing quality monitoring.

Direct analysis of silica gel extracts from immunostained glycosphingolipids by nanoelectrospray ionization quadrupole time-of-flight mass spectrometry.

Abstract: A combined strategy of preparative high-performance thin-layer chromatography overlay assay and mass spectrometry was established for the structural characterization of immunostained glycosphingolipids (GSLs) in silica gel extracts. Crude chloroform/methanol/water (30/60/8, v/v/v) extracts of immunostained TLC bands were analyzed by nanoelectrospray low-energy CID mass spectrometry without further purification. The GSL species investigated were isomeric monosialogangliosides of the neolacto series from a ganglioside preparation of human granulocytes, the disialoganglioside GD3 from a human melanoma lipid extract, and ganglio series Gg3Cer of a neutral GSL preparation from murine lymphoreticular MDAY-D2 cells. For the specific detection of lipid-bound oligosaccharides, polyclonal chicken IgY, murine monoclonal IgG3, and IgM antibodies were used. The resulting mass spectra show that only analytical quantities of approximately 1 microg of a single GSL within a complex mixture are required for the structure determination of immunostained GSLs by MS and MS/MS. All species investigated were detected as singly charged deprotonated molecular ions, and neither buffer-derived salt adducts nor coextracted contaminants from the immunostaining procedure or the silica gel layer were observed. This effective HPTLC-MS-joined procedure offers a wide range of applications for any carbohydrate binding agents such as bacterial toxins, plant lectins, and others.

Differentiation of black gel inks using optical and chemical techniques.

Abstract: Gel ink pens have become a common writing instrument in the United States. Questioned document examiners often attempt to optically differentiate gel inks from each other and from other non-ballpoint ink writings (e.g., those from roller-ball pens). Since early formulations were primarily pigment-based, they do not elute when analyzed by thin-layer chromatography. However, recent gel ink formulations (i.e., within the past five years) include dye-based inks that can be easily separated. This study differentiates black gel inks using optical and chemical techniques. The techniques include: microscopy, visible and near infrared reflectance, near infrared luminescence, thin-layer chromatography (TLC), spot tests, and gas chromatography/mass spectrometry (GC/MS). As a result of this study a flow chart has been developed allowing for a systematic determination of a questioned ink. In addition, an analysis of volatile compounds found in gel inks revealed that there are some unique ingredients that may be found in gel inks that are not typically found in other non-ballpoint inks.

The determination and quantification of photosynthetic pigments by reverse phase high-performance liquid chromatography, thin-layer chromatography, and spectrophotometry.

Abstract: Chorophylls and carotenoids are functionally important pigment molecules in photosynthetic organisms. Methods for the determination of chlorophylls a and b, beta-carotene, neoxanthin, and the pigments that are involved in photoprotective cycles such as the xanthophylls are discussed. These cycles involve the reversible de-epoxidation of violaxanthin into antheraxanthin and zeaxanthin, as well as the reversible de-epoxidation of lutein-5,6-epoxide into lutein. This chapter describes pigment extraction procedures from higher plants and green algae. Methods for the determination and quantification using high-performance liquid chromatograpy (HPLC) are described as well as methods for the separation and purification of pigments for use as standards using thin-layer chromatography (TLC). In addition, several spectrophotometric methods for the quantification of chlorophylls a and b are described.

Determination of the mycotoxin fumonisin B1 in maize by reversed-phase thin-layer chromatography: a collaborative study.

Abstract: A simple and cost-effective method using thin-layer chromatography for the determination of the mycotoxin fumonisin B1 in maize is described. The analytical method consisted of the extraction of ground maize by shaking with methanol/water (75:25) for 60 min and clean-up of the resultant extract by means of strong anion exchange solid-phase extraction. The purified residue, formed by evaporation of the elution solvent, was reacted with fluorescamine and the fumonisin B1-derivative was separated by reversed-phase thin-layer chromatography using a developing solution of methanol/aqueous 4% potassium chloride (70:30). The derivatized FB1 was readily visualized as a greenish-yellow spot under long wavelength ultraviolet light and quantified by visual comparison with a set of similarly derivatized standards in the range 20-300 ng FB1 spotted on plate. Based on visual comparison, levels down to 0.5 mg kg(-1) were successfully estimated. The method was collaboratively studied in 14 laboratories using four duplicate maize meal samples (including a blank) and a spiked sample for determination of recovery. No significant difference was observed between mean FB1 levels by high-performance liquid chromatography or thin-layer chromatography. Based on within-laboratory relative standard deviations of 27.1-41.7% and between-laboratory relative standard deviations of 35.0-63.3%, the method can be considered semiquantitative. The mean recovery achieved by participants at a spiking level of 2.00 mg kg(-1) was 74.5%.

Lipid classes, fatty acid composition and triacylglycerol molecular species in the kernels of pumpkin (Cucurbita spp) seeds

Abstract: The lipids extracted from the kernels of pumpkin (Cucurbita spp) seeds of three cultivars were classified by thin layer chromatography into six fractions: steryl esters (SEs, 0.5–1.2%), triacylglycerols (TAGs, 92.7–93.4%), free fatty acids (FFAs, 2.9–3.5%), sn-1,3-diacylglycerols (1,3-DAGs, 0.4–0.9%), sn-1,2-diacylglycerols (1,2-DAGs, 0.7–0.9%) and phospholipids (PLs, 1.5%). Fatty acids derivatised as methyl esters were analysed by gas chromatography with flame ionisation detection. Molecular species and fatty acid distributions of TAGs, isolated from the total lipids in the kernels, were analysed by a combination of argentation thin layer chromatography (TLC) and gas chromatography. A modified argentation TLC procedure, developed to optimise the separation of the complex mixture of total TAGs, provided 11 different groups of TAGs, based on both the degree of unsaturation and the total chain length of fatty acid groups. With a few exceptions, SM2 (5.8–20.1%), S2D (8.8–11.2%), M3 (6.7–24.8%), SMD (6.8–16.7%), M2D (16.7–23.6%), SD2 (4.6–15.1%) and MD2 (4.9–18.6%) were the main TAG components. These results suggest that there are significant differences (P < 0.05) not only in fatty acid distributions of acyl lipids but also in molecular species of TAGs among the three cultivars. The differences in pumpkin cultivars could be appreciable, based on the distribution of molecular species in TAGs. However, pumpkin seed kernels could be utilised successfully as a source of edible oils for human consumption. Copyright © 2004 Society of Chemical Industry

Development of a reversed-phase thin-layer chromatographic method for artemisinin and its derivatives.

Abstract: In this study a clear separation between seven analogues of artemisinin on thin-layer chromatography (TLC) is presented. The developed TLC method is carried out on a RP-C18 thin-layer plate using acetonitrile-water (50:25 v/v) as the mobile phase. Spots are visualized by derivatization with an acidified 4-methoxybenzaldehyde reagent in methanol-water. This method allows the separation of a diverse group of compounds that have versatile hydrophilic/lipophilic characteristics; namely artemisinin, artesunate (AS), artelinic acid (AL), arteether (AE), both isomers of artemether (AM) (alpha and beta), dihydroartemisinin, and desoxyartemisinin. Separation of some degradation products and impurities, down to 2%, allows quality control and stability investigation of all actives in raw material and pharmaceutical formulations. The method is further developed via densitometric measurement for quantitative determination purposes for AL and AS. The derivatization technique is evaluated, showing good stability and reproducibility of the coloring process. Percent relative standard deviation values are less than 5% for replicates, and linearity is obtained in the range of 0.5 to 8 microg. A comparative study with high-performance liquid chromatography (HPLC) on a C18 column, applying the same mobile phase, proves the suitability of the TLC method, in which almost all presented analytes are separated from each other. In contrast, HPLC requires at least a 20-min analysis to chromatograph all of the compounds and only betaAM and AE are clearly separated from each other and from the other compounds.

Characterization of oligosaccharides in milk of bearded seal (Erignathus barbatus).

Abstract: Carbohydrates were extracted from milk of a bearded seal, Erignathus barbatus (Family Phocidae). Free neutral oligosaccharides were separated by gel filtration, anion-exchange chromatography and preparative thin layer chromatography, while free acidic oligosaccharides were separated by gel filtration and then purified by ion exchange chromatography, gel filtration and high performance liquid chromatography. Oligosaccharide structures were determined by H-NMR spectroscopy. The structures of the neutral oligosaccharides were as follows; lactose, 29-fucosyllactose, 1

Separation of a mixture of eighteen pesticides by two-dimensional thin-layer chromatography on a cyanopropyl-bonded polar stationary phase

Abstract: Eighteen pesticides have been separated by two-dimensional thin-layer chromatography on a moderate polarity CN-modified silica gel. CN-silica is widely used as a adsorbent in both normal- and reversed-phase chromatography. Large selectivity differences are obtained by combination of both NP and RP modes on cyanopropyl-bonded polar adsorbents. The greatest spread of points was obtained by combining nonaqueous normal-phase mobile phases (tetrahydrofuran or ethyl acetate in n -heptane) and aqueous reversed phases (a polar solvent (methanol or acetonitrile) in water), both on thin layers of cyanopropyl-bonded polar adsorbent. Correlations of R F values in NP and RP systems were used for practical separation of a mixture of eighteen pesticides by 2D TLC on this adsorbent. Plates were scanned and videoscanned to show the real pictures of the 2D TLC separation.

Phospholipids of Clostridium perfringens: a reexamination

Abstract: We have identified phosphatidylethanolamine as one of the major phospholipids of Clostridium perfringens by two dimensional thin layer chromatography of the intact lipids and of their deacylation products and by liquid chromatography followed by mass spectrometry of the intact neutral phospholipid fraction. The principal fatty acids of phosphatidylethanolamine are myristic acid (14:0), lauric acid (12:0), and palmitic acid (16:0) and the major molecular species are 14:0,14:0 (26.3%); 12:0,14:0 (19.0%); 14:0,16:0 (22.4%) and 16:0,16:0 (17.6%). A similar distribution of molecular species was found in the other major phospholipid, O-alanyl phosphatidylglycerol.

A new reagent for identification of amino acids on thin-layer chromatography plates

Abstract: A variety of selective and non-selective reagents [1–18] are used in thin-layer chromatography for identification of amino acids. Such identification is very useful because of the occurrence of the amino acids as the structural units of proteins, in the free state in natural products, and for determination of the C-terminal amino acids of degraded proteins. Of the reagents generally used, ninhydrin, a non-selective reagent, is popular because of its remarkably high sensitivity [2], even though it produces the same purple color with all the amino acids except proline and hydroxyproline. To solve this problem a new reagent, 4hydroxyacetophenone–isatin-5-sulfonic acid (sodium salt) is proposed. This communication deals with this reagent, which produces distinguishable colors for most of the amino acids. With the proposed reagent detection limits for the amino acids range between 0.1 and 2.0 μg.

Transdermal iontophoresis of insulin: III. Influence of electronic parameters

Abstract: Transdermal iontophoresis is a physical enhancement strategy primarily for charged molecules and offers a number of advantages for the delivery of peptides and proteins. The singular advantage of iontophoresis lies in the precise control of dose by manipulating the current protocol. The objective of the present investigation was to understand the role of electronic parameters on iontophoretic transport of large peptides using insulin as a model peptide. Ex vivo permeation experiments were conducted using excised rat skin and the influence of varying current strengths, duration, on/off ratios and switching iontophoresis on insulin permeation were studied. High performance liquid chromatography (HPLC), polyacrylamide gel electrophoresis (SDS-PAGE) and thin layer chromatography (TLC) were used to assess the electrochemical stability of insulin; while Fourier transform infra-red (FT-IR) spectroscopy and thermogravimetric analysis (TGA) were used to understand the biophysical changes in skin during iontophoresis. The permeation of insulin was found to increase as a function of current strength and duration of current application. Skin barrier integrity and electrochemical stability of insulin was dependent on the charge applied during iontophoresis. FT-IR spectroscopy and TGA studies showed that the skin hydration increased with increase in the charge applied and thus facilitated the transport of insulin. Periodic iontophoresis did not show any significant difference in insulin permeation compared with continuous current application; 1:1 on/off ratio resulted in higher amount of insulin permeation, while flux was highest with mixed duty cycle. Switching iontophoresis was useful in reducing the pH shift and in improving the electrochemical stability of insulin at pH 3.6 and 7.4, respectively. The electroosmotic flow was influenced by the pH of the donor medium, as well as by the electrode polarity during switching and non-switching iontophoresis. Overall, the study demonstrates the issues related to the optimization of electronic parameters for the iontophoretic delivery of a large peptide.

Evaluation of the Lipophilicity of Some Dehydroepiandrosterone Derivates Using RP-18 HPTLC Chromatography

Abstract: The chromatographic behavior of DHEA derivates - 17a-substituted-3b,17b-dihydroxy-16oximino derivatives of 5-androstene has been studied by reversed-phase high-performance thin-layer chromatography. HPTLC was performed on a C-18 bonded phase with two aqueous eluents, acetone-water and dioxane-water. Linear relationship between the retention parameters (RM) and the organic modifier content in the mobile phase allows the extrapolation procedure. The influence of substituent in the molecule on extrapolated retention data is discussed. The correlation between the chromatographic lipophilic parameters (RM 0 ) and the calculated log P values, as well as biological activity, has been studied. The results show that reversedphase chromatographic (RM 0 ) are useful in describing the lipophilic nature of investigated compounds as well as the activity.

Analysis of Phenolic Components in Croatian Red Wines by Thin-Layer Chromatography

Abstract: A thin-layer chromatographic (TLC) method has been used for determination of phenolic compounds in two samples of wine commercially available in Croatia. Sample preparation was by liquid-liquid extraction with diethyl ether at pH 2.0. Extracts and standard solutions were applied to 20 cm × 20 cm silica gel 60 F 254 TLC plates. After treatment of the developed plates with ammonia vapor, phenolic compounds were detected on chromatograms by their colors or by fluorescence in UV light at λ = 244 nm and at λ = 366 nm before and after spraying with 1% ethanolic AlCl 3 . The efficiency of eleven mobile phases was tested by three mathematical techniques — calculation of the information content ( I ) derived from Shannon’s equation, determination of discriminating power ( DP ), and formation of clusters and dendrogram. It was shown that the best mobile phase for separation of phenolic compounds in the wine extracts was benzene-ethyl acetate-formic acid, 30 + 15 + 5 ( v / v ). Six phenolic compounds were identified...

System for analysis of explosives

Abstract: A system for analysis of explosives. Samples are spotted on a thin layer chromatography plate. Multi-component explosives standards are spotted on the thin layer chromatography plate. The thin layer chromatography plate is dipped in a solvent mixture and chromatography is allowed to proceed. The thin layer chromatography plate is dipped in reagent 1. The thin layer chromatography plate is heated. The thin layer chromatography plate is dipped in reagent 2.