Continuous Thin-layer Chromatography Research Articles

Review and Proposal of Alternative Technologies for Comprehensive and Reliable Newborn Screening Using Paper Borne Urine Samples for Lysosomal Storage Disorders: Glycosphingolipid Disorders

Abstract Few current methods are efficient to detect a high number of lysosomal storage disorders (LSDs) in newborn screening. Therefore, we propose a stepwise procedure that starts with the use of paper borne urine samples (Berry-Woolf specimen) for the inexpensive detection of elevated lysosomal content and the identification of which of the three majors biochemical groups -mucopolysaccharides, oligosaccharides, and glycosphingolipids- is detected. Urine samples are preferable to blood samples because of their higher concentrations of the relevant analytes. Subsequent steps would precisely determine which enzyme deficiency is involved. As a summary, following our previous papers on the detection of elevated oligosaccharides and mucopolysaccharides, here we describe how elevated urinary glycosphingolipids (GSLs) could be fluorometrically detected using the reagent 5-hydroxy-1-tetralone (HOT) and subsequently identified with precision by continuous thin layer chromatography or other techniques. We also outline the steps required for the validation of this procedure for its introduction in newborn screening programs.

Monoclonal Antibody AA4, Which Inhibits Binding of IgE to High Affinity Receptors on Rat Basophilic Leukemia Cells, Binds to Novel α-Galactosyl Derivatives of Ganglioside GD1b

Abstract Mouse monoclonal antibody AA4 inhibits the binding of IgE to high affinity IgE receptors on the rat basophilic leukemia cell line RBL-2H3. As shown by immunostaining of thin layer chromatograms, antibody AA4 binds avidly to two disialogangliosides (antigen I and antigen II) that occur in this cell line. The two antigens were purified by anion exchange chromatography followed by short-bed continuous thin-layer chromatography. About 230 micrograms of antigen I and 60 micrograms of antigen II were obtained from 20 g (wet weight) of leukemia cells. The structures of both purified antigens were determined to be alpha-galactosyl derivatives of the ganglioside GD1b by fast atom bombardment-mass spectrometry, by chemical ionization-mass spectrometry of permethylated samples, by gas chromatography-mass spectrometry of partially methylated alditol acetates, and by treatment with exoglycosidases and mild acid hydrolysis. The structure of antigen I is: (formula; see text) Antigen II has an additional alpha-galactosyl residue as follows: (formula; see text) The ceramide of antigen I contains approximately equal amounts of C24:0, C22:0, C20:0, C18:0, and C16:0 N-acyl fatty acids. The ceramide base is predominantly sphingosine along with a small amount of dihydrosphingosine. In contrast, the ceramide of antigen II contains mainly C24:0 N-acyl fatty acid with much lower amounts of C22:0, C20:0, and C18:0 fatty acids. Moreover, the ceramide base is approximately 55% sphingosine and 45% dihydrosphingosine. No unsaturated N-acyl fatty acids were detected in either antigen.

Computer simulation and optimization of preparative thin-layer chromatography under conditions of isocratic and stepwise gradient elution

An equilibrium sandwich chamber for continuous thin-layer chromatography was used to study overloaded systems. Mixtures of two or three dyes were used as the model samples, and wide starting zones were formed (volume-overloaded systems). The movement of the zones was recorded during continuous elution. The effect of sample volume and the mode of development (isocratic or gradient) on the maximum separation yield was investigated. A computer program was developed for the calculation of the final RF values for the front and rear edges of the bands.

A Simple Device for Gradient Elution in Equilibrium Sandwich Chambers for Continuous Thin-Layer Chromatography

Abstract The separation efficiency of various thin-layer chromatographic techniques was investigated using a horizontal sandwich tank for continuous elution with a glass distributer. Complex extract of camomile flowers (Azulane) was used as the test mixture and the number of separated spots as the measure of efficiency. Best results were obtained when gradient elution with chloroform-ethyl acetate mixtures was applied, A simple miniaturized device without mechanical mixer was employed, the spontaneous mixing being enhanced by gravitational flow of the components and their association on the molecular level.

Formation and migration of zones in overloaded preparative liquid-solid chromatography

An equilibrium sandwich chamber for continuous thinlayer chromatography was used to study overloaded systems of the heptane + methylene chloride-silica type. Mixtures of two or three dyes were used as the model samples. Wide starting zones were formed using a glass distributor (frontal chromatography stage), then the movement of the zones was recorded during continuous elution. The effect of sample concentration and volume on the maximum separation yield was investigated. Band compression effects are illustrated for samples dissolved in solvents having a low eluent strength. Satisfactory analogy was found to separations in preparative column chromatography. Good separation yield was obtained for frontal + elution TLC: depending on the differences in the RF values of the components, 6–16 mg samples were completely separated on 0.5×100×75 mm layers containing ca. 1 g of silica.

The Separation of Dichlorobenzophenone Isomers by Continuous Development and Reversed-Phase Thin Layer Chromatography

Abstract Two thin-layer chromatographic methods are described for 2,4'-dichlorobenzophenone with which 1% of the 2,2'-and 2,3'-isomers can be determined. Continuous thin-layer chromatography on silica gel and conventional reversed-phase thin-layer chroma-tography were found to be complementary techniques for the separation of the dichlorobenzophenone isomers.

Solvent demixing effects in continuous thin-layer chromatography and their elimination

Demixing effects in thin-layer chromatography have been investigated for frequently used binary solvents of various compositions, and methods of eliminating such effects are described. The simplest way of eliminating solvent demixing consists in presaturation of the plate in the vapours of the polar solvent component or pre-development with its more concentrated solution. The sample should be spotted some distance behind the front of the developing solvent to secure steady-state conditions analogous to column development with an equilibrated mobile phase. For more concentrated polar solvents (e.g., ⪢ 20%) it is sufficient to spot the sample several centimetres behind the solvent front; the RF values of solvent demixing are then high so that the presaturation or predevelopment step can be omitted. Procedures for the elimination of solvent demixing for typical solvent mixtures in the minimum time are given in terms of the distance of predevelopment.

Simple device for continuous thin-layer chromatography

Simple device for continuous thin-layer chromatography