Separation of glycoprotein-derived oligosaccharides by thin-layer chromatography.

A thin-layer chromatography (tlc) system has been developed for the separation of glycoprotein-derivedoligosaccharides. The method involves chromatography on silica gel using n-propanol/acetic acid/water (3:3:2 v/v) as the solvent. This tlc method was used to separate pathological oligosaccharides isolated from individuals with G_M1 gangliosidosis and with neuraminidase deficiency. The results indicate the potential usefulness of the system in the analysis of complex carbohydrates.     

Separation of sterols by vapor-programmed thin-layer chromatography

By vapor-programmed thin-layer chromatography on silica gel, it was possible to separate cholestanol from cholesterol and stigmastanol from beta-sitosterol. The method was applied for the analysis of beta-sitosterol-3-(14)C.     

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.     

Separation by thin-layer chromatography and structure elucidation of bilirubin conjugates isolated from dog bile.

1. A system for separation of bile pigments by t.l.c. and for their structure elucidation is presented. Separated bile pigments are characterized by t.l.c. of derived dipyrrolic azopigments. 2. At the tetrapyrrolic stage hydrolysis in strongly alkaline medium followed by t.l.c. demonstrates the presence of bilirubin-IIIalpha, -IXalpha and -XIIIalpha and allows assessment of their relative amounts. 3. Most structural information is derived from analysis of dipyrrolic azopigments. Such derivatives, obtained by treatment of separated bile pigments with diazotized ethyl anthranilate, were separated and purified by t.l.c. Micro methods showed (a) the nature of the dipyrrolic aglycone, (b) the nature of the bonds connecting aglycone to a conjugating group, (c) the ratio of vinyl/isovinyl isomers present in the aglycone and, (d) the nature of the conjugating groups (by suitable derivative formation and t.l.c. with reference to known compounds). 4. In bile of normal dogs at least 20 tetrapyrrolic, diazo-positive bile pigments could be recognized. Except for two pigments the tetrapyrrolic nucleus corresponded predominantly to bilirubin-IXalpha. All conjugated pigments had their conjugating groups connected in ester linkage to the tetrapyrrolic aglycone, Apart from bilirubin-IXalpha, monoconjugates and homogeneous and mixed diconjugates of bilirubin were demonstrated; conjugating groups of major importance were xylose, glucose and glucuronic acid. 5. Bilirubin isomer determination on native bile and isolated bile pigments, and dipyrrole-exchange assays with [14C8]bilirubin indicated (a) that the conjugates pre-exist in bile, and (b) that no significant dipyrrole exchange occurs during isolation of the pigments.     

Separation of bile acids of rat bile by thin-layer chromatography

The common bile acids of rat bile (chenodeoxycholic, hyodeoxycholic, cholic, alpha-muricholic, and beta-muricholic acids) are completely separated by a new thin-layer chromatographic system.     

Separation of sterol acetates by column and thin-layer argentation chromatography

Column and thin-layer chromatographic systems employing silver nitrate-impregnated adsorbents are described for the separation of sterol acetates which differ in the number of double bonds in the steroid nucleus or side chain.     

Separation of gluco- and galactocerebrosides by means of borate thin-layer chromatography

Gluco- and galactocerebrosides can be separated by thin-layer chromatography on Silica Gel G prepared with sodium borate solution instead of water. The most successful developing system was chloroform-methanol-water-15 M NH(4)OH 280:70:6:1.     

Separation of phosphatidylinositols and other phospholipids by two-step one-dimensional thin-layer chromatography

A quick and efficient thin-layer chromatographic procedure is described for the separation of phosphatidylinositol-4,5-bisphosphate, phosphatidylinositol-4-monophosphate, phosphatidylinositol, phosphatidylcholine, phosphatidic acid, and phosphatidylethanolamine. The method involves development of the phospholipids successively in two different solvent systems but in the same direction. The method is simple, reproducible, and gives good resolution of the six different phospholipids tested. About 8-10 32P-labeled phospholipids isolated from rat hepatocytes were separated by this method; the six mentioned above were identified. Thus, the technique has potential application for both qualitative and quantitative analysis of phospholipid mixtures, such as the phosphoinositides, in cell or tissue extracts.     

Separation of proline and hydroxyproline using thin-layer chromatography

Thin-layer chromatography allows rapid separation of proline and hydroxyproline over a wide quantitative range.The present method utilizes the nitrous acid reaction to destroy amino acids in protein hydrolyzates. After ether extraction of amino acid derivatives, proline and hydroxyproline are present unchanged or as unstable n-nitroso derivatives and can be rapidly separated by thin-layer chromatography on cellulose. Mixtures including up to 500 μg of each imino acid are separable on a 250-μ thick layer. The imino acids can be eluted and determined colorimetrically. Studies using isotopically labeled proline and hydroxyproline are possible even when low specific activities are involved.This simple and rapid technique has proved useful in determination of the specific activity of radioactive protein bound hydroxyproline in crude collagen extracts.     

Separation of oligonucleotides by thin-layer electrophoresis.

The relative reaction rates of various hydrolases such as cellulase, xylanase, protease, amylase, and pectinase are readily determined using an oscillating capillary tube recording viscometer. The continuous readout allows initial reaction rates to be observed. The complete reaction curve of these enzymes was found to approximate a rectangular hyperbola. This finding allowed the calculation of the equation of each curve and the extrapolation of the initial reaction rates.