Polyamide Thin-layer Chromatography Research Articles

Improvement researches on quality standard of Anemarrhenae Rhizoma and its raw processed products

This study is to improve the quality standard and supply the scientific basis for Anemarrhenae Rhizoma and its raw processed products. Steroidal saponin including timosaponin BⅡ, timosaponin AⅢ and flavonoids including neomangiferin and mangiferin were selected as the indicative components. Silica gel G thin layer chromatography(TLC) and polyamide TLC were used to detect the two types of compounds, respectively. The contents of timosaponin BⅡ and timosaponin AⅢ were determined by HPLC-ELSD and the content of neomangiferin, mangiferin and isomangiferin were determined by HPLC-UV. Moisture, total ash and acid insoluble ash were determined according to Chinese Pharmacopoeia(2015 edition). And 80% ethanol was selected as the solvent and the content determination of total extract were determined. The fingerprints of Anemarrhenae Rhizoma and its raw processed products were established by HPLC-UV and HPLC-ELSD. The results showed that the methods of TLC and HPLC have been successfully stablished. There are 2 and 3 peaks which have been identified by HPLC-ELSD and HPLC-UV, respectively. The HPLC fingerprint methods are specific and can be used to identify and quality control for Anemarrhenae Rhizoma and its raw processed products in the mass. Comparing to Chinese Pharmacopoeia(2015 edition), the TLC identification and content determination were revised and the total extract determination and HPLC fingerprints were added in the present study. Our results can be used as the scientific basis of quqlity control for Anemarrhenae Rhizoma and its raw processed products.

Separation of catechins and O-methylated (-)-epigallocatechin gallate using polyamide thin-layer chromatography.

Thin-layer chromatography (TLC) method for the separation and quantitative determination of seven related compounds: (+)-catechin (C), (-)-epicatechin (EC), (-)-epigallocatechin (EGC), (-)-epicatechin gallate (ECG), (-)-epigallocatechin gallate (EGCG), (-)-epigallocatechin-3-O-(3-O-methyl) gallate (EGCG3″Me) and (-)-epigallocatechin- 3-O-(4-O-methyl) gallate (EGCG4″Me) has been developed. The above-mentioned seven compounds have been resolved using polyamide TLC plates using a double-development with methanol followed by acetone/acetic acid (2:1, v/v). In addition, separation of the phenolic acids namely gallic acid, chlorogenic acid, and caffeic acid was achieved using the same solvent system. The applicability of the method was checked by screening of extracts of green, black, oolong, white tea and tea cultivars leaves.

A Rapid and Green Limit Test Method for Five Synthetic Colorants in Foods Using Polyamide Thin-layer Chromatography

Synthetic food colorants are used in very small amounts. Typically, the colorants are extracted, purified and concentrated prior to analysis; however, sample preparation is time-consuming, of high cost and environmentally unfriendly. In this work, a polyamide thin-layer chromatographic (TLC) method in combination with on-plate solid-phase extraction and backlight-assisted detection has been developed for five commonly used synthetic colorants in foods. Beverages (degassed where appropriate) were directly applied to the polyamide TLC plate. Gelatinous and solid samples were first extracted with water–ammonia aqueous solution (25 %) (99:1) assisted by ultrasonic treatment. Sample solutions were then acidified before application. Relatively large volumes of solution (5–25 μl) could be applied to the plate without significant sample diffusion. Before development, the start spots were cleaned up using ethanol (95 %)–water–acetic acid (5:5:0.1). The plate was developed using a mobile phase of ethanol (95 %)–water–ammonia aqueous solution (25 %) (75:14:1). After development, the plate was viewed in backlight mode with an LED array light. The selectivity of the method proved acceptable. The limits of detection for Ponceau 4R, Amaranth, Tartrazine, Sunset Yellow and Brilliant Blue were 10.1, 9.59, 4.19, 4.12 and 2.06 ng, respectively. The method was rapid, low-cost and sensitive, providing a green limit test method for five colorants in foods.

Analysis of flavones in Rubus parvifolius Linn by high performance liquid chromatography combined with electrospray ionization-mass spectrometry and thin-layer chromatography combined with Fourier transform surface enhanced Raman spectroscopy

Qualitative and quantitative analyses of total flavones in wild Rubus parvifolius Linn (RPL) were carried out using ultraviolet spectroscopy, and the isolation and structure determination of the main active ingredients were carried out by high performance liquid chromatography (HPLC) coupled with Electrospray ionization-mass Spectroscopy (ESI-MS), thin layer chromatography (TLC), and the Fourier transform surface enhanced Raman spectroscopy (FT-SERS). Two flavones were isolated from RPL and they were identified as herahydroxy dihydroflavanonol and quercetin. Two chromatographic spots were obtained when polyamide TLC plate was used to separate the flavones, and two strengthen bases were prepared by the reduction of silver nitrate with trisodium citrate and also by the photo-induced reduction of a mixture of silver nitrate and potassium bromide. Strong increase of characteristic Raman scatting intensity could be observed on these bases for microgram quantity of the flavones compound.

Effect of Chamber Gas Phase on Mobile Phase pH and on Separation Efficiency in TLC. A New Mode of Chromatography

A new variant of thin-layer chromatography (TLC), based on a gradual change of mobile phase acidity during elution, is proposed. The pH change occurs in the mobile phase moving along the TLC plate as a result of its contact with an acidic or a basic gas phase that replaces the initial mobile phase vapor in the TLC chamber. The potential of this approach has been demonstrated by using carbon dioxide and ammonia gases to improve the resolution of benzoic acids and aromatic amines on polyamide TLC plates.

High-resolution analysis of catechol-type siderophores using polyamide thin layer chromatography

The iron-deficient culture supernatant of a soil bacterial strain identified as Erwinia sp. was analyzed using a new high-resolution polyamide thin layer chromatography (TLC) and a silica TLC. The results showed both TLC methods were very effective for separating simple catechol compounds such as 2,3-dihydroxybenzoic acid (2,3-DHBA) and catechol. However, in the analysis of more complicated catechol compounds or true catechol-type siderophores (conjugates of 2,3-DHBA and amino acids), the polyamide TLC had the higher resolution. Polyamide TLC analysis showed that strain S1 produced three distinct catechol-type siderophores.

TLC Fractionation and Characterization of Ames Mutagenic Substances in Chlorine-treated 4-methylphenol Solution in the Presence of Bromide Ion

The diethyl ether extracts from aqueous 4-methylphenol solutions in the presence of bromide ion after treatment with chlorine were mutagenic to the Ames salmonella test strain TA100 in the absence of rat liver homogenate. Gas chromatography/mass spectrometry (GC/MS) showed the occurrence of halogenated products in the extracts: bromo-4-methylphenols, bromo-4-methylquinones and brominated 4-methylphenol dimers. The diethyl ether extracts were fractionated into several fractions by polyamide thin-layer chromatography (TLC) . The fractionated components were examined for mutagenicity by means of Ames assays, and were identified by GC/MS. TLC fraction of the extracts revealed that two components present in the extracts are mutagenic. GC/MS analysis indicated the presence of 2, 6-dibromo-4-hydroxy-4-methylcyclohexa-2, 5-lien-1-one (2, 6-dibromotolquinol) as the major mutagen and brominated 4-methylphenol dimers as minor mutagenic compounds in the chlorinated 4-methylphenol solutions containing bromide ion. Production of 2, 6-dibromotolquinol in water was found to be dependent on the equivalents of chlorine per mole of compound and the reaction pH.

TLC-Fractionation and GC/MS Characterization of Mutagenic Substances in Chlorinated 4-Alkylphenol Solutions.

The diethyl ether extracts from aqueous 4-alkylphenol solutions after treatment with chlorine were mutagenic to the Ames Salmonella tester strains TA98 and TA100 in the absence of rat liver homogenate. Gas chromatography/mass spectrometry (GC/MS) showed the occurrence of chlorinated products in the extracts; chloro-4-alkylphenols, chloro-4-alkylquinones and chlorinated 4-alkylphenol dimers. The diethyl ether extracts were fractionated into several fractions by polyamide thin-layer chromatography (TLC) . The fractionated components were examined for mutagenicity by means of Ames assay, and were identified by GC/MS. TLC fractionation of the extracts revealed that two components present in the extracts are mutagenic. GC/MS analyses indicated the presence of 2, 6 -dichloro-4-hydroxy-4-alkylcyclohexa-2, 5-dien-1-ones as the major mutagen and chlorinated 4-alkylphenol dimers as minor mutagenic compounds in the chlorinated 4-alkylphenol solutions.

Thin-layer chromatographic fractionation of Ames mutagenic chlorosemiquinone in chlorinated 4-methylphenol solution

Abstract The diethyl ether extract from an aqueous solution of 4-methylphenol after treatment with chlorine was mutagenic to the Ames Salmonella test strain TA100 in the absence of liver homogenate. Gas chromatography-mass spectrometry (GC-MS) showed the occurrence of chlorinated products in the extract: chloro-4-methylphenols, chloro-4-methylquinones and chlorinated 4-methylphenol dimers. The diethyl ether extract was fractionated into several fractions by polyamide thin-layer chromatography (TLC). The fractionated components were examined for mutagenicity by means of Ames assays, and were identified by GC-MS. TLC fractionation of the extract revealed that two components present in the extract are mutagenic. GC-MS analysis indicated the presence of 2,6-dichloro-4-hydroxy-4-methylcyclohexa-2,5-dien-1-one as the major mutagen and chlorinated 4-methylphenol dimers as minor mutagenic compounds in the chlorinated 4-methylphenol solution.

Rapid Analysis of the Food Colors in Foodstuffs Using Polyamide

A rapid and simple analysis procedure involving a batch system using polyamide (nylon-6 powder) was devised for the detection of food colors. The food colors were extracted from food with a mixture of 1% ammonia water and ethanol (4:6). The extract was filtered, and the filtrate was diluted two or three times with water. The colors in the solution were absorbed on a small amount of polyamide (10-30mg) in acid (pH 4-5). After filtration and rinsing with hot water, the polyamide was suspended and eluted with a small quantity (about 100μl) of a mixture of 28% ammonia water and ethanol (4:6). After five min, the supernatant was used for paper chromatography and thin layer chromatography on a silica gel plate or a polyamide sheet. The solvent systems for the chromatographies were as follows: paper chromatography, butanol-ethanol-1% ammonia water (6:2:3); silica gell thin layer chromatography, ethyl acetate-ethanol-28% ammonia water (3.3:1:1) or (4.5:1:0.7); polyamide thin layer chromatography, methanol-ethanol-isoamyl alcohol-28% ammonia water (15:10:5:3). The systems containing ammonia as described above were required for separation of the food colors. Compared with the wool dyeing method, the presented method did not require evaporation of alcohol, heating for adsorption or elution of the food colors or concentration of the eluate. It is rapid, simple and precise for the detection of the food colors in various colored foods.

Comparative Studies on Tree Pollen Allergens

The previously isolated major allergen of birch pollen (fraction BV45), Int. Archs Allergy appl. Immun. <i>68</i>: 70–78 (1982), was further purified by recycling chromatography. The purified preparation was run on a high-performance liquid chromatography (HPLC) TSK-G-2000 gel filtration chromatography column and, finally, on paper high-volt electrophoresis. The protein recovered met the homogeneity criteria required for performing the N-terminal sequence analysis. The allergenic and antigenic reactivities of the HPLC-purified protein, designated BV45B, was examined. A single homogeneous precipitation line in crossed immunoelectrophoresis (CIE) was shown. Specific IgE-inhibition tests and immuno-autoradiographic prints indicated that this allergen could bind reaginic IgE specificially and with good affinity. The homogeneity of BV45B was examined by isoelectric focusing (IEF). Several minor bands of pI differences of less than 0.1 units were visible, demonstrating the existence of some molecular variants of this protein. The N-terminal sequence analysis of the molecule was performed, and the following four amino acids were tentatively shown by sequential cleavage: NH<sub>2</sub>-Ala-Gly-Ile-Val-. The demonstration of one dominant N-terminal l-dimethyl-amino-5-naphthalene sulphonyl (DNS)-amino acid by polyamide thin-layer chromatography at each sequence step confirmed that (a) the N-terminal residue of the protein was not blocked; (b) the heterogeneity shown by the IEF system was merely due to the presence of several homologous polymorphic proteins with identical N-terminal amino acid, (c) the adequacy of the purification repertoire used.

Chemical changes of organic compounds in chlorinated water : XI. Thin-layer chromatographic fractionation of ames mutagenic compounds in chlorine-treated 4-methylphenol solution

The diethyl ether extract from an aqueous solution of 4-methylphenol after treatment with hypochlorite was mutagenic to the Ames Salmonella test strain TA100 in the absence of liver homogenate. Gas chromatography—mass spectrometry (GC—MS) showed the occurrence of, at least, twenty compounds in the extract: chloro-4-methylphenols, chlorohydroxy-4-methylquinones and chlorinated 4-methylphenol dimers. The diethyl ether extract was fractionated into several fractions by silica gel and polyamide thin-layer chromatography (TLC). The fractionated components were then examined for mutagenicity by means of Ames assays, and were identified by GC—MS. TLC fractionation of the extract revealed that the major components present in the extract are not mutagenic, but minor components (less than 4% of the total extract) are mutagenic. GC—MS analysis indicated the presence of chlorinated 4-methylphenol dimers in the fraction which exhibited the highest mutagenicity.

Comparative Studies on Tree Pollen Allergens

The previously isolated major allergen of birch pollen (fraction BV45), Int. Archs Allergy appl. Immun. 68: 70-78 (1982), was further purified by recycling chromatography. The purified preparation was run on a high-performance liquid chromatography (HPLC) TSK-G-2000 gel filtration chromatography column and, finally, on paper high-volt electrophoresis. The protein recovered met the homogeneity criteria required for performing the N-terminal sequence analysis. The allergenic and antigenic reactivities of the HPLC-purified protein, designated BV45B, was examined. A single homogeneous precipitation line in crossed immunoelectrophoresis (CIE) was shown. Specific IgE-inhibition tests and immuno-autoradiographic prints indicated that this allergen could bind reaginic IgE specificially and with good affinity. The homogeneity of BV45B was examined by isoelectric focusing (IEF). Several minor bands of pI differences of less than 0.1 units were visible, demonstrating the existence of some molecular variants of this protein. The N-terminal sequence analysis of the molecule was performed, and the following four amino acids were tentatively shown by sequential cleavage: NH2-Ala-Gly-Ile-Val-. The demonstration of one dominant N-terminal 1-dimethyl-amino-5-naphthalene sulphonyl (DNS)-amino acid by polyamide thin-layer chromatography at each sequence step confirmed that the N-terminal residue of the protein was not blocked; the heterogeneity shown by the IEF system was merely due to the presence of several homologous polymorphic proteins with identical N-terminal amino acid, the adequacy of the purification repertoire used.

Polyamide thin-layer chromatography of phosphorylated tyrosine, threonine, and serine

One-dimensional thin-layer chromatography on polyamide plates offers an easy and rapid identification of O-phosphotyrosine. The thin-layer plate is developed for 30 min in 5% propionic acid containing 0.013%–0.025% sodium dodecyl sulfate. O-Phosphotyrosine, with R f = 0.54, can be well separated from O-phosphothreonine and O-phosphoserine, which comigrate at R f = 0.72.

Separation of azaarenes by polyamide thin-layer chromatography and the relationship between their basicity and R F values

The separation of quinoline, isoquinoline, their methyl derivatives and the isomers of benzoquinolines and dibenzoquinolines was investigated by thin-layer chromatography using polyamide as adsorbent. When developed with neutral solvent systems, derivatives and isomers of azaarenes constructed of the same number of aromatic rings were hardly separated. However, using n-hexane—ethyl acetate—methanol—formic acid systems, separation was achieved due to differences in the basicity of the molecules caused by the effect of methyl groups and aromatic rings on the nitrogen atoms.

By-products as an aid in residue identification during peptide sequence analysis with dimethylaminoazobenzene isothiocyanate

4-N,N-Dimethylaminoazobenzene 4′-isothiocyanate degradations permit sensitive and fast manual sequence analysis, but assignments of some residues are difficult. Hydrophobic residues, especially leucine and isoleucine, are badly resolved on polyamide thin-layer chromatography. Differently colored by-products have been described before for a few labile residues, but it is now shown that most residues can give rise to characteristic by-products. These have different colors and chromatographic properties, depending on the nature of the parent residue. Thus, two to three sets of spots characterize each residue, giving multiple identification with increased reliability. Although variable and dependent on chemicals and conditions, by-products are often prominent after conversion with 50% trifluoroacetic acid, and can be utilized to improve the identifications.

Crosslinkage analysis of bacterial cell walls using dual-isotope labeling of N-terminal groups with fluorodinitrobenzene: A method of general application for microdetermination of free amino groups in peptides

An isotope-dilution procedure for quantitative N-terminal microanalysis using 3H-labeled 1-fluoro-2,4-dinitrobenzene and a mixture of free and acetylated 14C-labeled amino acids as internal standards is described. The 2,4-dinitrophenyl derivatives are separated by polyamide thin-layer chromatography. Although primarily intended for the estimation of crosslinkage in bacterial cell wall peptidoglycan, the method is capable of extension to other peptides. When tested on lysozyme, the procedure gave results which were in good agreement with accepted values.

Separation of dinitrophenyl derivatives of amino sugars and aminodeoxyalditols by polyamide thin-layer chromatography

The separation of glucosamine, galactosamine, muramic acid and the corresponding aminodeoxyalditols as their 2,4-dinitrophenyl (DNP) derivatives by two- dimensional thin-layer chromatography on polyamide is described. Reaction conditions are recommendation that minimize artefact formation during derivatisation. Initial development with 2% (v/v) acqueous formic acid separates the DNP derivatives from salts and by-products, which otherwise interfere with separation. Solvents for the second dimension contain chlorobenzene and acetic acid, with ethylenediamine- tetra-acetic acid to reduce tailing. Inclusion of benzeneboronic acid is particularly effective for separating compounds of interest.

Detection of Some Natural Dyes by Polyamide Thin-Layer Chromatography

Detection of Some Natural Dyes by Polyamide Thin-Layer Chromatography

Improved Procedure for the Estimation of Nanogram Quantities of Indole-3-acetic Acid in Plant Extracts using the Indolo-α-pyrone Fluorescence Method

The indolo-alpha-pyrone fluorescence method of determining indole-3-acetic acid (IAA) is improved by adding butylated hydroxytoluene (BHT), an antioxidant, to samples: addition of BHT increases the fluorescence intensities and decreases their variability so that amounts of IAA as small as 0.1 to 1 nanogram become measurable. Interfering compounds, 4-chloroindole-3-acetic acid and 5-hydroxyindole-3-acetic acid, can be separated from IAA by thin-layer chromatography using polyamide as the solid support, and benzene-ethyl acetate-acetic acid (70:25:5, v/v) as the developing solvent. Polyamide thin-layer chromatography is also superior in purifying IAA without significant loss or decomposition.A serious problem in extracting IAA from plant tissues is the considerable loss. Losses can result from decomposition of IAA when its solutions in organic solvents are evaporated to dryness, apparently due to some impurity in the solvents. Decomposition can be eliminated by simple treatments of the solvents, i.e. addition of BHT, washing with water, and passage through cotton wool. The latter is particularly effective in the case of ethyl acetate and ether. Loss of IAA by photodecomposition is also substantial even in dried samples. Ways to minimize the conversion of indole-3-pyruvic acid to IAA during extraction procedure are also described.A revised procedure of IAA extraction and determination is proposed. Applied to etiolated shoots of maize seedlings, it enables content to be estimated (18 nanograms per gram fresh weight) with over 90% recovery in terms of [(14)C]IAA added at the beginning of the extraction.