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Two-dimensional thin layer chromatography on cyano-bonded polar stationary phase was performed to optimize the separation of some antioxidant phenolic compounds from Eupatorium cannabinum extracts. Propan-2-ol mixed with n-heptane and ethyl acetate mixed with n-heptane were used as non-aqueous mobile phases in normal phase separations (1st direction of development in 2-dimensional high-performance thin-layer chromatography (2D-HPTLC mode)) and methanol mixed with water was used as a mobile phase in reversed phase (2nd direction of development in 2D-HPTLC mode). The plates were sprayed by use of Merck TLC sprayer using 2-(diphenylboryoxy)-ethylamine and PEG4000 (Merck, Darmstadt, Germany) or DPPH and photographed in CAMAG Cabinet UV lamp at 254 nm and 365 nm by use of Fuji 8 mpx camera. The plots RF non-aqueous mobile phase vs R F aqueous mobile phase were prepared to find optimal orthogonal 2D-TLC systems for the separation of investigated test compounds and then extracts from Eupatorium cannabinum. Satisfactory separations of antioxidant phenolic compounds in E. cannabinum extracts were obtained by use of optimized 2D-HPTLC systems.

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A two-dimensional thin-layer chromatographic fingerprint has been developed on a polyamide plate for the quality control of Helleborus thibetanus Franch. The investigated sample was separated by chloroform-ethyl acetate-methanol (3.0:8.0:4.4, v/v) in the first dimension and isooctane-n-propyl alcohol-water (10:2.5:1.0, v/v) adding 0.28 mol L−1 sodium dodecyl sulfate, a reversed micelle, in the second dimension. The plate was dried in the air at room temperature and examined in ultraviolet (UV) light at λ = 365 nm after development. The two developments were carried out over a distance of 70 mm. The two-dimensional thin-layer chromatographic method was validated in terms of repeatability, stability, and robustness. For fingerprint analysis, nine spots were identified as common spots. Statistical method (canonical correlation analysis) was first used to calculate the degree of similarity between the two-dimensional chromatograms. The proposed method was novel and accurate for the identification and quality evaluation of H. thibetanus Franch.

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For very complex multicomponent mixtures, it is preferable to use different variants of multidimensional (planar) chromatography. In multidimensional separations, the modes of planar chromatography commonly applied are comprehensive 2D planar chromatography on mono and bilayers, coupled-layer chromatography, combination of multidimensional planar chromatographic (MDPC) techniques, and also hyphenated methods. Different multidimensional (planar) chromatography techniques and hyphenated methods may be alternative modes for separation and identifcation of pesticides in environmental analysis, especially after coupling with modern detection techniques. Proper identifcation and quantitative analysis of the analytes are possible through the use of sensitive detection techniques such as high-resolution mass spectrometry (HRMS).

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. (1978) Microanalysis of brain lipids: multiple two-dimensional thin-layer chromatography. J. Lipid. Res. 19 , 916-921. Microanalysis of brain lipids: multiple two-dimensional thin-layer chromatography

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