Spray detection of phospholipids on thin-layer chromatograms

A new spray reagent with copper, ammonium molybdate, and sulphuric acid for the detection of phospholipids on thin-layer chromatograms is described. The specificity and sensitivity of the spray reagent is also shown.     

Modification of the Dittmer-Lester reagent for the detection of phospholipid derivatives on thin-layer chromatograms.

A simple modification of the Dittmer-Lester reagent is described that allow the detection of phospholipid derivatives at very low concentrations on silica gel and reversed-phase thin-layer plates. This modification, which involves the addition of acetic acid to the mixture, permits the observation of sharp blue spots on a white background. The specificity and sensitivity of the spray are discussed.     

Simple scraper for thin-layer chromatograms

A simple scraper for rapid and quantitative transfer of zones on thin-layer chromatograms to liquid scintillation counting vials is described.     

Immunostaining free oligosaccharides directly on thin-layer chromatograms

Oligosaccharides are chromatographed on amino-bonded high-performance thin-layer chromatography silica gel plates and after chromatography the aldehydes on the reducing ends of the oligosaccharides react with the amino groups on the silica gel. Bound oligosaccharides are immunostained directly on the chromatograms by monoclonal antibodies. The binding of antibodies is detected by autoradiography after a second incubation with 125I-labeled goat anti-mouse immunoglobulin. Using this method, 10 pmol of lacto-N-difucopentaose I (Leb hapten) and lacto-N-fucopentaose III (Lex hapten) are detected directly on the thin-layer chromatograms by monoclonal antibodies 10c17 and 534F8, respectively. Previously undescribed larger oligosaccharides containing these epitopes are also detected in human milk. This method may be used to identify and characterize antibody-binding oligosaccharides liberated from glycoconjugates by hydrazinolysis, by trifluoroacetolysis, by ozonolysis, or by treatment with endoglycosidases. This technique may also be used to determine the structural specificity of other carbohydrate-binding proteins such as lectins, toxins, and hormones or of bacteria and viruses that bind to cell surface glycoproteins.     

A procedure for detecting phosphonolipids on thin-layer chromatograms

A simple modification of a phospholipid-specific spray for thin-layer chromatograms allows it to be used as a specific detection reagent for phosphonolipids and as a general lipid detection reagent.     

Short bed-continuous development thin-layer chromatography of glycosphingolipids

The technique of short bed--continuous development chromatography has been utilized to increase the separation of glycosphingolipids on high performance thin-layer chromatography plates. The theoretical goal of increasing separation of bands by decreasing solvent strength was achieved within a practical time span as a result of the high solvent velocities in the short bed tank. Examples are given for increased separation of short and long chain neutral glycolipids, acetylated neutral glycolipids, and gangliosides.     

Permanent sensitive stain for choline-containing phospholipids on thin-layer chromatograms

The phosphomolybdic blue stain for choline of Levine and Chargaff can be used for detection of small amounts of choline-containing phospholipids on thin-layer chromatograms if they are first fixed with polyvinyl propionate to permit a washing step.     

Phosphorus determination in phosphoglycerides from thin-layer chromatograms

Phosphoglyceride phosphorus is readily determined directly on silica gel removed from thin-layer chromatograms, without elution, by a nondigestive procedure with a sulfuric-periodic acid reagent. The method is specific for hydrolyzable phosphoglyceride containing two acid-hydrolyzable groups. Cariolipin reacts only partially, while sphingomyelin, diether lecithin, and phosphonate cephalin fail completely to react.     

Specific staining on thin-layer chromatograms of glycosphingolipids of neolacto series and gangliosides with a terminal N-acetylneuraminyl residue by different procedures with wheat germ agglutinin

Sensitive staining methods with wheat germ agglutinin were developed for the detection of glycosphingolipids of neolacto series (A) and gangliosides with a terminal N-acetylneuraminyl residue (B) on thin-layer chromatograms. (A) Neolacto series glycosphingolipids were treated by beta-galactosidase on the chromatograms in the presence of taurodeoxycholate. Then the chromatograms were incubated with biotinated wheat germ agglutinin followed by incubation with a complex of avidin and biotinated horseradish peroxidase, and the reaction was detected by 4-chloro-1-naphthol. In the case of gangliosides, sialidase treatment on the chromatograms was performed before the beta-galactosidase treatment. The sensitivity of the method for Lc3Cer, nLc4Cer, sialyl-nLc4Cer, and sialyl-nLc6Cer was 4 pmol, 7.6 pmol, 2.9 pmol and 1.4 pmol, respectively. (B) The gangliosides on the chromatograms were oxidized by periodic acid and reduced by NaBH4. Then the chromatograms were stained with wheat germ agglutinin as mentioned above. As little as 0.5 pmol of GM3, NeuAc-nLc4Cer, and NeuAc-nLc6Cer was detected by this method, whereas the detected limits for these gangliosides were 10 pmol, 10 pmol and 2 pmol, respectively, when periodate oxidation was omitted. GM4, GD3 and GD1a were an order less reactive than GM3, GM2, GM1 or GD1b were not stained under the same condition. In contrast to NeuAc-containing gangliosides, any gangliosides with N-glycolylneuraminic acid were not stained by the method in (B).     

Chromatography of sugars in body fluids : III. Stepwise detection of sugars with aniline citrate on paper and thin-layer chromatograms

The paper chromatographic detection method of White and Hess for urinary aldoses using aniline citrate has been studied and modified. As the reactivity of individual sugars is determined mainly by their molecular weight and structure, our modification is based on a gradual increase in the reaction temperature (room temperature, 37 or 55 and 105°) and variations in heating time. This technique enables reaction conditions to be obtained that lead to a full display of both the transient and final characteristics of the fluorescence and colour of the individual spots in the sugar spectrum of any analyzed material.Compared with other reagents based on aniline or other aromatic amines, aniline citrate possesses unusually wide operational flexibility. The procedure is suitable for paper and thin-layer chromatography and is especially valuable in the analysis of urinary and tissue sugar extracts for the identification of largely overlapping spots with different reaction times, the differentiation of oligosaccharides, particularly those with different linkages, and obtaining additional data for the identification of unknown metabolites.     

The use of 1,6-diphenylhexatriene to detect lipids on thin-layer chromatograms

1,6-diphenylhexatriene (DPH) fluoresces violet under long-wave uv light in the presence of lipid on silica gel thin-layer chromatograms. The DPH, sprayed as a solution in a volatile nonpolar solvent, detects submicrogram quantities of all lipid classes. It is a nondestructive marker and can be removed subsequently by elution with a petroleum ether (40–60°C).     

Semiquantitative estimation of aflatoxins on thin-layer chromatograms with a gray scale

The disappearance of the fluorescence of aflatoxin spots on TL chromatograms by zones with different shades of gray of a Kodak gray scale is the basis of a technique for the semiquantitative estimation of aflatoxins. Standard curves for B₁ and G₁ are given.     

Immunostaining on thin-layer chromatograms of oligosaccharides released from gangliosides by endoglycoceramidase

A method for immobilizing oligosaccharides on a TLC plate for immunostaining has been developed. N-Glycolylneuraminic acid (NeuGc)-containing oligosaccharides derived from II3NeuGc-LacCer, IV3NeuGc-nLcOse4Cer, II3NeuGc-GgOse3Cer, and II3(NeuGc)2-LacCer by digestion with our newly isolated endoglycoceramidase (Ito, M. & Yamagata, T. (1986) J. Biol. Chem. 261, 14278-14282) and sialyllactose were chromatographed on polyamide 11 TLC or NH2-HPTLC plates, and covalently linked to the plates by reductive amination with sodium cyanoborohydride (NaBH3-CN). The immobilized oligosaccharides were detected by enzyme-immunostaining using NeuGc-specific chicken anti-NeuGc-LacCer and horseradish peroxidase-conjugated rabbit anti-chicken IgG. II3NeuGc-nLcOse4 showed the highest reactivity with the antibody, followed by II3NeuGc-GgOse3. As little as 0.8 nmol of the NeuGc-containing oligosaccharides was detected. The polyamide 11 TLC aluminum plate was found to be more suitable for the immunostaining than the NH2-HPTLC plate under the conditions used. For binding of the oligosaccharides to the NH2-HPTLC plate, reductive amination was found to be superior to the heating method reported earlier.     

Separation of neutral glycosphingolipids and sulfatides by thin-layer chromatography

Two one-dimensional systems for separation of glycolipids from total lipid extracts of tissues by thin-layer chromatography are described. System I used, as adsorbent, an alkaline mixture of silica gel without CaSO(4) binder (75%) and magnesium silicate (25%), and the lipids were "developed" with three successive solvent mixtures. The separated compounds (from the fastest to the slowest moving) were: ceramide, ceramide monohexosides, sulfatides, ceramide dihexosides, psychosine, ceramide trihexosides, and ceramide N-acetylhexosamine trihexosides. In system II a two-step development was used on an adsorbent consisting of silica gel without CaSO(4) binder (80%) and magnesium silicate (20%). The separated compounds were: ceramides, ceramide monohexosides, and ceramide dihexosides. Psychosine and sulfatides as well as ceramide trihexosides and ceramide N-acetylhexosamine trihexosides were not separated. In both systems all neutral lipids moved to the very top of the chromatogram and phospholipids stayed at the origin. Application of systems I and II for separation of glycolipids was demonstrated on total lipid extracts from animal tissues.