Thin-layer Chromatography of Triose Reductone and its Relative Compounds

Application of thin-layer chromatographic techniques to the analysis and preparation of triose reductone from naturally occuring reductone compounds has become an important tool in reductone chemistry. A satisfactory method for the separation of triose reductone and related compounds by thin-layer chromatography, using silica gel plate and various solvents as developer, is described. Seven reductones were separated from each other by two-dimensional chromatography.     

Analysis of metabolism of carcinogenic polycyclic hydrocarbons by position-sensing proportional counting of thin-layer chromatograms.

A procedure which combines thin-layer chromatography with position-sensing proportional counting has been developed for analyzing the metabolism of carcinogenic polycyclic aromatic hydrocarbons. The profiles of the metabolites of [³H]benzo(a)pyrene and 7,12-[12-¹⁴C]dimethylbenz(a)anthracene produced in cell culture were comparable when obtained by this procedure and by standard methods. However, position-sensing proportional counting allows simultaneous counting of all components of a sample within 10–20 min, and thereby permits the analysis of many hydrocarbon samples in a short time. In addition, the procedure eliminates the necessity of cutting or scraping carcinogen-containing thin-layer chromatograms.     

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 Rf = 0.54, can be well separated from O-phosphothreonine and O-phosphoserine, which comigrate at Rf = 0.72.     

Determination of peptidylglycine alpha-amidating monooxygenase activity in human serum by thin-layer chromatography

We developed a simple assay system for the quantitative evaluation of peptidylglycine alpha-amidating monooxygenase activity using as substrate a 125I-labeled synthetic tripeptide, 125I-D-Tyr-Val-Gly, thin-layer chromatography, and a radiochromatoscanner. The basic principle of this method is that thin-layer chromatography separates the reaction product, 125I-D-Tyr-Val-NH2, from the substrate in an assay mixture. The 125I activities of both substrate and product separated from each other on a thin-layer chromatography plate were quantified with a radiochromatoscanner and the rate of conversion of the substrate to the product was calculated from their counts. Human serum was used as an enzyme source and the values of alpha-amidation activity obtained by our method under optimal conditions were almost identical to those of the published method using ion-exchange chromatography (sulphopropyl-Sephadex C-50 column) and a gamma-counter. Our method makes it possible to estimate the 10-pmol level of the product using 10 microliters of human serum and to assay a large number of samples rapidly and easily. It is therefore thought to be very useful for screening various tissues for alpha-amidation activity.     

Separation of methylated free bile acids from their taurine and methyl glycine conjugates by thin-layer chromatography

Class separation of methylated free bile acids from bile acids conjugated with taurine and methylglycine was accomplished using a solvent system of 2,2,4-trimethylpentane-absolute ethanol 10:1 (v/v). By developing a silica thin-layer plate two times with solvent in a Brinkmann sandwich tank, the difficult resolution between methyl cholate and methyl glycolithocholate was achieved. Evidence is presented that this separation system may be useful as a preparative step in the analysis of bile acids by gas-liquid chromatography or high pressure liquid chromatography.--Bolt, M. J. G. Separation of methylated free bile acids from their taurine and methyl glycine conjugates by thin-layer chromatography.     

Gradient-thickness thin-layer chromatography for the isolation and analysis of trace amounts of free fatty acids in large lipid samples

A thin-layer chromatographic method for quantitative isolation of free fatty acids is described. This method appears to be more satisfactory than existing methods in offering the combination of advantages of specificity, simplicity, rapidity, reproducibility, accuracy, high sensitivity, and applicability as a preparative technique. The method involves chromatography on a thin-layer plate on which the layer of Silica Gel G decreases linearly in thickness from 1000 micro at the base to 125 micro at the upper end. This gradient-thickness design allows the separation and densitometric quantitation of very small traces of free fatty acids from relatively large and complex lipid samples in a single chromatographic step. The method has been shown to be applicable directly to the crude total lipid extracts of several mammalian tissues. It appears to generate little if any artifactual free fatty acids from the breakdown of complex lipids, in contrast to the undesirable behavior of silicic acid columns in this respect. Gradient-thickness thin-layer chromatography promises to be useful for the quantitative isolation of trace amounts not only of other types of lipids but also of classes of compounds other than lipids.     

High-performance thin-layer chromatographic separation of ranitidine hydrochloride and two related compounds

Ranitidine hydrochloride and its two related compounds, used in the USP TLC purity testing of the drug, were separated on a high-performance thin-layer chromatography (HPTLC) RP-18 WF_254S precoated plate using methanol–3% NH₄OH (4:1, v/v) as the mobile phase. The main advantage of the proposed HPTLC system over the USP TLC system for testing the purity of ranitidine is a better and more efficient separation of these three compounds in a shorter time and with less consumption of solvents. The system is promising from the point of view of the development of a new method for the TLC purity testing of ranitidine hydrochloride. A video system was used for imaging thin-layer chromatograms. Direct UV densitometric quantitation of the three compounds and a model for the calculation of analytical performance parameters is presented in the second part of the paper.     

Simple screening method for molds producing intracellular mycotoxins in pure cultures.

A simple screening method for molds producing the intracellular mycotoxins brevianamide A, citreoviridin, cyclopiazonic acid, luteoskyrin, penitrem A, roquefortine C, sterigmatocystin, verruculogen, viomellein, and xanthomegnin was developed. After removing an agar plug from the mold culture, the mycelium on the plug is wetted with a drop of methanol-chloroform (1:2). By this treatment the intracellular mycotoxins are extracted within seconds and transferred directly to a thin-layer chromatography plate by immediately placing the plug on the plate while the mycelium is still wet. After removal of the plug, known thin-layer chromatographic procedures are carried out. The substrate (Czapek yeast autolysate agar) and growth conditions (25 degrees C for 7 days) used by Penicillium taxonomists proved suitable for the production of the mycotoxins investigated when 60 known toxigenic isolates and 865 cultures isolated from foods and feedstuffs were tested with this screening method.     

Determination of amitriptyline and nortriptyline in human plasma by quantitative thin-layer chromatography

A thin-layer chromatographic method for simultaneous determination of amitriptyline (AT) and nortriptyline (NT) in human plasma is described. Both substances are extracted from biological material by means of a single extraction. The extract is evaporated until dry and the residue quantitatively applied to a silica gel thin-layer plate. AT and NT are separated from interfering plasma components by chromatography. The spots are visualized by nitration, reduction and coupling with N-(1-naphtyl)ethylenediamine on the plate. The intensity of the azo-dyes formed can be measured densitometrically. Using 1 ml of plasma, the sensitivity limit was 0.5 ng/ml for both substances. About 10–15 plasma samples can be analysed per day. The method is applicable to pharmacokinetic studies after a single oral dose of 25 mg AT as hydrochloride in man.     

2',3'-cyclic nucleotide phosphohydrolase activity determined using an image analyzer detection system

The activity of 2',3'-cyclic nucleotide phosphohydrolase (CNPase) was assayed using high-performance thin-layer chromatography (HPTLC) and an image analyzer detection system. The assay system was used to study a possible inhibitory effect by aminoguanidine on CNPase specific activity. One advantage of using a fixed-time HPTLC system over a real-time spectrophotometric system for an enzyme activity study was that apparent inhibition of the enzyme due to interference of the assay system (chromophore inhibition, etc.) was avoided. In addition, due to the increased accuracy of the image analyzer over conventional methods of TLC plate analysis, a rapid and more accurate measurement of HPTLC plates was possible which required only nanomole amounts of substrate. Also, a digital image of each plate analyzed was stored indefinitely in the computer's memory for future reference. The measurements of CNPase specific activity made using this system compared favorably to those found in recent literature.     

Synthesis of diadenosine 5',5'-P1,P4-tetraphosphate (AP4A) in sea urchin embryos

Sea urchin embryos were labeled with [3H]adenosine at two developmental stages (morula and prism) and the labeled acid-soluble nucleotides were fractionated successively by column chromatography with DEAE-Sephadex and DEAE-cellulose, and by thin-layer chromatography on a PEI-cellulose plate. Significant radioactivity was detected on the PEI-cellulose plate at the region of diadenosine 5',5'-P1,P4-tetraphosphate (AP4A). After treatment of this fraction with phosphodiesterase, the radioactivity was all recovered in the AMP region, while alkaline phosphatase had no effect on the AP4A fraction. The present result suggests that AP4A is actively synthesized in the sea urchin embryos.     

In situ immunological determination of basic carbohydrate structures of gangliosides on thin-layer plates

An immunological method for the determination of the basic carbohydrate structure of gangliosides by using a thin-layer chromatographic immunostaining technique was developed. After high-performance thin-layer chromatography of gangliosides, the chromatogram is treated with a 0.4% polyisobutylmethacrylate solution. Arthrobacter ureafaciens neuraminidase is then applied to the separated gangliosides in situ on the chromatographic plate. This procedure will remove both external and internal sialic acid residues from the core oligosaccharide backbone. The resulting glycolipid products are then incubated with anti-Gg4 serum and 125I-staphylococcal protein A, successively, and exposed to an X-ray film. Through a highly specific binding, the anti-Gg4 antibody detects only those gangliosides having the oligosaccharide backbone of Gg4.     

A rapid and sensitive method for the determination of the lithogenic index of bile by thin-layer chromatography and densitometry using a dual-wavelength chromatoscanner

A rapid and sensitive method for quantitative determination of three bile lipid components, cholesterol, bile acids, and lecithin, is described. These components were separated by thin-layer chromatography on a silica gel plate, spots were visualized with a phosphomolybdate reagent, and their color intensities were estimated by direct densitometry using a dualwavelength chromatoscanner. The lithogenic index was estimated by the molar ratio of the three lipids. This method can be applied to the routine analysis of bile in patients with gallstones. It has been evaluated by comparison with the method using standard gas-liquid chromatography and spectrophotometry.     

Separation of individual sulfated bile acid conjugates as calcium complexes using reversed-phase partition thin-layer chromatography.

A method for separating individual monosulfated primary bile acid conjugates by reversed-phase partition thin-layer chromatography on octadecyl-bonded silica gel is described. The solvent system is acetonitrile containing calcium, probably as calcium carbamate. Excellent resolution of the 3- and 7-monosulfated glycine conjugates, as well as 3- and 7-monosulfated taurine conjugates of cholic and chenodeoxycholic acids is reported. A convenient class separation of sulfated from nonsulfated primary bile acid conjugates by adsorption thin-layer chromatography on low-polarity silica gel is also described.     

Oxalate-silica gel thin-layer system for free 2-hydroxy fatty acids and for fatty acyl coenzyme A

The 2-hydroxy fatty acids tend to yield streaks in thin-layer chromatography on silica gel plates. If potassium oxalate is included with binder-free silica gel, good spots are obtained. Similar difficulties are found in paper chromatography of the fatty acid derivatives of coenzyme A, especially with long-chain acids. The same thin-layer system gives good spots with these compounds.     

Thin-layer chromatography blotting for the fluorescence detection of phospholipid hydroperoxides and cholesteryl ester hydroperoxides

A blotting technique was developed to specifically detect lipid hydroperoxides in thin-layer chromatography. Phosphatidylcholine hydroperoxides and cholesteryl linoleate hydroperoxides ranging from 0.1 to 0.5 nmol, which were prepared by reaction with soybean lipoxygenase, were visualized as fluorescent spots on the blotted membrane by immersing the plate into a blotting solvent containing 0.01% (w/v) diphenyl-1-pyrenylphosphine. This technique was applied successfully to monitor lipid peroxidation in human low-density lipoprotein in vitro.     

A microdetermination of ( 14 C)lactate in biological fluids

A simple method, based on a single-step conversion of lactate to pyruvic acid semicarbazone by lactic dehydrogenase, has been developed for the microdetermination of [¹⁴C]lactate in the biological fluids. Pyruvic semicarbazone thus formed is efficiently separated from other labelled metabolites by means of charcoal treatment followed by thin-layer chromatography on cellulose plate. The entire procedure applied to rat whole blood revealed the mean recovery of 75.2% with a good reproducibility.     

Silica-immobilized His6-tagged enzyme: alanine racemase in hydrophobic solvent

A new immobilization method for enzymes is presented to facilitate synthetic applications in aqueous as well as organic media. The enzyme Alanine racemase (AlaR) from Geobacillus stearothermophilus was cloned, overexpressed and then immobilized on a silica-coated thin-layer chromatography plate to create an enzyme surface. The enzyme, fused to a His(6)-tag at its N-terminal, was tethered to the chemically modified silica-coated TLC plate through cobalt ions. The immobilized enzyme showed unaltered kinetic parameters in small-scale stirred reactions and retained its activity after rinsing, drying, freezing or immersion in n-hexane. This practical method is a first step towards a general immobilization method for synthesis applications with any enzyme suitable for His6-tagging.     

Direct analysis of glycolipids on thin-layer plates by matrix-assisted secondary ion mass spectrometry: application for glycolipid storage disorders

The lipids accumulated in organs of patients with Gaucher's, Tay-Sachs, and Fabry's disease were identified by means of the combination of thin-layer chromatography and matrix-assisted secondary ion mass spectrometry. The total lipid extract of each lipidosis tissue was chromatographed on a TLC plate and then analyzed directly by mass spectrometry without elution of the sample from the TLC plate. The amount of material needed to obtain an adequate spectrum is in the order of a few micrograms of lipids per band for both positive and negative ion detection. By scanning the plates, mass spectral and chromatographic information can be obtained simultaneously, which was shown to be useful for the qualitative identification of the components on the plates.     

Lipid A fractions analyzed by a technique involving thin-layer chromatography and enzyme-linked immunosorbent assay

A modified enzyme-linked immunosorbent assay (ELISA), with alkaline phosphatase as enzyme, was used for the study of antigenicity of lipid A fractions directly on thin-layer chromatographic (TLC) plates. For visualization a gel slab containing the enzyme substrate was placed on the plate containing enzyme-conjugated antibodies. The plate was read by a thin-layer chromatogram spectrophotometer. The immunoassay was both highly specific and quite sensitive. Sensitivity was superior to levels obtained by staining the plate with molybdenum blue (for phosphate) or orcinol (for carbohydrate). Fractions of lipid A from Escherichia coli 0111, Shigella flexneri or Salmonella minnesota R595, after being separated by thin-layer chromatography, were analyzed using rabbit anti-(lipid A) serum. Patterns obtained by scanning the same plates for phosphate staining and for the TLC-ELISA corresponded well. For comparison with TLC-ELISA, an inhibition assay was run using a tube ELISA. The tube ELISA, run in aqueous medium, showed that fractions 6-8 (those having the highest RF values) had the least activities. In contrast, TLC-ELISA did not detect large differences between fractions 2-7. This discrepancy probably reflected limited aqueous solubility of fractions 6 and 7. We conclude that TLC-ELISA might reveal antigenic activities of lipids that could be missed by other methods. The data suggested that all fractions, except for fraction 8, were similar in their antigenicity by TLC-ELISA. Differences in antigenicity between the fractions occurred when the fractions were tested in free form in an aqueous environment and these differences possibly could have been due to different solubilities of individual fractions.