Fluorescence detection by intensity changes for high-performance thin-layer chromatography separation of lipids using automated multiple development

Changes in emission of berberine cation, induced by non-covalent interactions with lipids on silica gel plates, can be used for detecting and quantifying lipids using fluorescence scanning densitometry in HPTLC analysis. This procedure, referred to as fluorescence detection by intensity changes (FDIC) has been used here in combination with automated multiple development (HPTLC/AMD), a gradient-based separation HPTLC technique, for separating, detecting and quantifying lipids from different families. Three different HPTLC/AMD gradient schemes have been developed for separating: neutral lipid families and steryl glycosides; different sphingolipids; and sphingosine-sphinganine mixtures. Fluorescent molar responses of studied lipids, and differences in response among different lipid families have been rationalized in the light of a previously proposed model of FDIC response, which is based on ion-induced dipole interactions between the fluorophore and the analyte. Likewise, computational calculations using molecular mechanics have also been a complementary useful tool to explain high FDIC responses of cholesteryl and steryl-derivatives, and moderate responses of sphingolipids. An explanation for the high FDIC response of cholesterol, whose limit of detection (LOD) is 5 ng, has been proposed. Advantages and limitations of FDIC application have also been discussed.     

径向展开薄层色谱法分离生物碱及其分离效能

径向展开薄层色谱法是一种将样品由中心沿径向向外展开的简便、快速、高效的色谱方法。该文组装了简单的径向展开薄层色谱装置,并建立了朱砂安神丸的径向展开薄层色谱检测法,对其中的生物碱成分进行分离,研究了径向展开薄层色谱的分离特性。从薄层色谱基础理论出发,对径向展开薄层色谱和一般薄层色谱的分离效能进行了比对研究,设计试验进行计算和求解。证明了径向展开薄层色谱法更快、更高效、更经济,适用于生物碱等高极性样品分离。探索了径向展开薄层色谱法高分离效率的理论根源,这一研究思路也为理论创新提供了新的方法和思路。     

Separation and identification of aliphatic amine derivatives with a new fluorescent reagent using high-performance liquid chromatography and thin-layer chromatography

Summary The use of 1,2-naphthoylenebenzimidazole-6-sulphochloride has been proposed as the reagent for derivatization of aliphatic amines prior to their separation, identification and quantitation both in HPLC and in TLC. The reaction of amines with this compound is quantitative and highly fluorescent derivatives are formed that provide favourable detection limits and sensitivity as compared to Dansyl derivatives of aliphatic amines. Actual detection limits achieved correspond to ca. 10^–10 mol of the amine in a spot after elution from the thin-layer plate and to ca. 5·10^–14 mol of the amine in a sample volume of 10 l injected into the liquid chromatograph. The use of this derivatization reagent offers good potential for the analysis of trace amounts of amines in environmental samples and in biological material.Presented at the 14th International Symposium on Chromatography London, September, 1982     

Separation of DABS derivatives of amino acids by multiple gradient development (MGD) in thin-layer chromatography

Summary A mixture of 13 DABS-amino acids has been chromatographed on high-performance silica gel layers developed with eluents containing increasing concentrations of ethyl acetate in heptane + chloroform, using a modification of stepwise multiple development MGD described in a earlier paper. Densitograms were obtained at 485 nm. The MGD method was very efficient, separating all 13 DABS-amino acids, and rapid, owing to the use of a non-aqueous mobile phase.     

Automated multiple development thin layer chromatography for calystegines and their biosynthetic precursors

Automated multiple development thin layer chromatography (AMD-TLC) was used for separation of calystegines, a class of nortropane alkaloids, and of precursors of their biosynthesis. The calystegines differ in the number of hydroxyl groups at the nortropane ring system and in the substitution pattern. A combination of TLC methods allows the separation of the individual calystegines and separation of possible precursors of the biosynthesis. Solvent combination, development time, the number of development steps, the drying time between each run and the preconditioning parameters of the silica gel TLC plates were optimised. Limits of detection were evaluated for several detection reagents.     

Completed preparative separation of alkaloids from Cephaltaxus fortunine by step-pH-gradient high-speed counter-current chromatography

Cephalotaxine-type alkaloids are the anti-cancer components in twigs, leaves, roots and seeds of Cephalotaxus fortunine. It is very important to use the limited resource by finding an efficient purification technology of the alkaloids. Separation of cephalotaxine-type alkaloids in Cephalotaxus fortunine by step-pH-gradient high-speed counter-current chromatography (step-pH-gradient HSCCC) was studied in this paper. The step-pH-gradient HSCCC was performed on a HSCCC instrument equipped with a 400-mL column, using the upper phase of ethyl acetate–n-hexane–water, with added 0.01% trifluoroacetic acid (TFA) as stationary phase, and the lower phase of ethyl acetate–n-hexane–water, with added 2% NH₄OH, 0.2% NH₄OH and 0.05% TFA as mobile phase. For each separation, 800 mg of extract of cephalotaxine-type alkaloids was separated to yield 9.3 mg of drupacine, 15.9 mg of wilsonine, 130.4 mg of cephalotaxine, 64.8 mg of epi-wilsonine, 12.8 mg of fortunine and 35.6 mg of acetylcephalotaxine with purities 81.2%, 85.7%, 95.3%, 97.5%, 89.1% and 96.2%, respectively. The recovery of each alkaloid was more than 90%. The structures of the six alkaloids were identified by electrospray ionization mass spectrum (ESI-MS) and ¹H and ¹³C NMR.     

Separation and fluorodensitometric determination of some tropolone alkaloids by thin-layer chromatography

Chromatographia - The tropolone alkaloids colchicine, colchiceine, demecolcine, demecolceine, isocolchicine, isocolchiceine, N-deacetylcolchicine, N-deacetylcolchiceine,...     

Investigation of Symphytum cordatum alkaloids by liquid-liquid partitioning, thin-layer chromatography and liquid chromatography-ion-trap mass spectrometry

From the alkalised crude extract of Symphytum cordatum (L.) W.K. roots, pyrrolizidine alkaloids (PAs) were extracted as free tertiary bases and polar N-oxides in a merely one-step liquid-liquid partitioning (LLP) in separation funnel and subsequently pre-fractionated by preparative multiple-development (MD) thin-layer chromatography (TLC) on silica gel plates. In this way three alkaloid fractions of different polarities and retention on silica gel plates were obtained as: the most polar N-oxides of the highest retention, the tertiary bases of medium retention, and diesterified N-oxides of the lowest retention. The former fraction was reduced into free bases by sodium hydrosulfite and purified by LLP on Extrelut-NT3 cartridge. It was further analysed together with the two other fractions by high-performance liquid chromatography (HPLC)-ion-trap mass spectrometry with atmospheric pressure chemical ionization (APCI) interface on XTerra C₁₈ column using a gradient elution. Based on MSⁿ spectra, 18 various alkaloids have been tentatively determined for the first time in this plant as the following types of structure: echimidine-N-oxide (three diasteroisomers), 7-sarracinyl-9-viridiflorylretronecine (two diasteroisomers), echimidine (two diasteroisomers), lycopsamine (two diasteroisomers), dihydroechinatine-N-oxide, dihydroheliospathuline-N-oxide, lycopsamine-N-oxide (three diasteroisomers), 7-acetyllycopsamine-N-oxide, symphytine-N-oxide (two diasteroisomers) and 2″,3″-epoxyechiumine-N-oxide.     

Analysis of the cinchona alkaloids by liquid chromatography. Reversed-phase chromatography on octadecylsilyl columns

Summary The complete separation of the major cinchona alkaloids and their dihydro derivatives is achieved in less than 30 minutes, using a Hypersil 5m octadecylsilyl column in the reversed-phase mode. Excellent peak symmetry is obtained by incorporation of a long chain amine additive in the acidic mobile phase.     

Improved quantitative thin-layer chromatographic method for the separation of cobalamin derivatives

Summary A new quantitative thin-layer chromatographic method was developed for the separation and determination of cobalamin derivatives. A mixture of acetone, acetonitrile, isopropanol, diethylamine and aqueous ammonium hydroxide as the eluent and precoated Kieselgel 60 chromatoplate as the TLC plate was used. The evaluation of the separated spots was carried out by densitometry. The method can be used for the determination of cyanocobalamin in fermentatic mixture without any cleaning or concentration procedure as well as for the purity test of cyanocobalamin and hydroxocobalamin.     

Identification, separation, and quantitative analysis of derivatives of nucleosides and nucleotides by thin-layer chromatography on cellulose

Summary The thin-layer chromatography of nucleoside and nucleotide derivatives on cellulose has been carried out, and R _f values and conditions for the separation and quantitative analysis of these mixtures by this method have been given.Khimiya Frirodnykh Soedinenii, Vol. 1, No. 5, pp. 335–342, 1965     

Separation and mechanism elucidation for six structure-like matrine-type alkaloids by micellar liquid chromatography

A mixed micellar liquid chromatography (MLC) method, the mobile phase consisting of anionic surfactant SDS and nonionic surfactant Brij35, was firstly developed for the separation and determination of six structure-like matrine-type alkaloids, including matrine, oxymatrine, sophocarpine, oxysophocarpine, sophoridine, and oxysophoridine. The factors influencing the resolution of the six alkaloids were systematically investigated and optimized, including the micellar composition and concentration, column temperature, the type and amount of organic solvent, and the pH values in the mobile phases. Under the optimized separation conditions, the six matrine-type alkaloids could be easily isocratically eluted with a baseline separation within 22 min. Under the designated conditions (SDS concentration from 10 to 50 mM, Brij35 from 5 to 30 mM, pH 3 and 5% 1-propanol), the hydrophobic selectivity was negatively correlated with the concentration of Brij35 but not with SDS. The functional group selectivity of the carbonyl group, double bond, and diastereomers, all decreased with the increase in percentage of SDS in the mixed micellar phase, because the strong electrostatic force masks other molecular forces which can discriminate the retention of the analytes. Therefore, such a combination in surfactants of MLC is a powerful strategy to increase the selectivity by adjusting the balance among the various molecular interaction forces influencing analytes' retention. Finally, the developed method was successfully used to separate and determine the contents of main alkaloids in Sophora medicinal plants, S. flavescens Ait. In summary, the mixed MLC is a valuable approach to separate and determine the structure-like multi-component natural samples.     

A rapid separation of citrus carotenoids by thin-layer chromatography

Summary A rapid separation of citrus carotenoid pigments is achieved by successive thin-layer chromatographic separations on two different adsorbents. The first chromatography on silica gel G developed with the solvent system acetone-petroleum ether (30∶70) gives a preliminary fractionation into groups with different polarity (hydrocarbons, monols, diols and polyols). A further separation of each group into individual carotenoids is obtained by rechromatography on MgO-Kieselguhr (1∶1, w/w). The same solvent system is used, the acetone percentage being increased according to the polarity of each group (from 4% to 30%).     

Determination of an opiate pentapeptide by on-line reversed phase preconcentration and ligand exchange chromatography with spectrofluorometric detection

Summary In order to study the occurrence of the opiate pentapeptide -[D-Ala^2,4, Tyr⁵] CM-5-NH₂ in the 10^–9–10^–5 M concentration range, we have developed a new method consisting of extraction of the peptide from its buffered aqueous medium onto a reversed phase cartridge of 50 L void volume, followed by on-line injection onto a Cu(II) modified silica gel column. The acetonitrile-rich mobile phase allows detection of the natural fluorescence of the peptide at the picomole level.     

Application of high-performance thin-layer chromatography and automated multiple development for the identification and determination of pesticides in water.

The combination of high-performance thin-layer chromatography (HPTLC) with automated multiple development (AMD) allows full automation of the separation step. This provides both a separation efficiency that is considerably better than that in conventional TLC and reproducible gradient elution on the thin layer. Reliable identification of trace amounts of pesticides (e.g., 28 ppt of atrazine) in subsoil, surface and drinking water is clearly demonstrated by AMD-HPTLC using a polarity gradient based on dichloromethane. A second universal elution gradient with changed selectivity on which the substances have different relative migration distances, identification by the multi-wavelength response correlation and the option of recording UV spectra in situ constitute three independent methods for reliable and rapid verification. Using 100- instead of 200-microns HPTLC silica gel layers and reducing the running distance increments from 3 to 2 or 1 mm increase the sensitivity, the linearity and the speed of the method.