Multiple Thin-layer Chromatography Research Articles

Progress in Detection and Structural Characterization of Glycosphingolipids in Crude Lipid Extracts by Enzymatic Phospholipid Disintegration Combined with Thin-Layer Chromatography Immunodetection and IR-MALDI Mass Spectrometry

In order to proceed in detection and structural analysis of glycosphingolipids (GSLs) in crude lipid extracts, which still remains a challenge in glycosphingolipidomics, we developed a strategy to structurally characterize neutral GSLs in total lipid extracts prepared from in vitro propagated human monocytic THP-1 cells, which were used as a model cell line. The procedure divides into (1) extraction of total lipids from cellular material, (2) enzymatical disintegration of phospholipids by treatment of the crude lipid extract with phospholipase C, (3) subsequent multiple thin-layer chromatography (TLC) overlay detection of individual GSLs with a mixture of various anti-GSL antibodies, and (4) structural analysis of immunostained GSLs directly on the TLC plate using infrared matrix-assisted laser desorption/ionization orthogonal time-of-flight mass spectrometry (IR-MALDI-o-TOF MS) in combination with collision-induced dissociation (CID). Whereas GSLs were mostly undetectable in untreated crude lipid extracts, pretreatment with phospholipase C resulted in clear-cut mass spectra. MS(1) and MS(2) analysis gave similar results when compared to those obtained with a highly purified neutral GSL preparation of THP-1 cells, which served as a control. We could demonstrate in this study the feasibility of simultaneous multiple immunodetection of individual neutral GSLs in one and the same TLC run and their structural characterization in crude lipid extracts after phospholipase C treatment, thereby avoiding laborious and long-lasting sample purification. This powerful combinatorial technique allows for efficient structural characterization of GSLs in small tissue samples and takes a step forward in the emerging field of glycosphingolipidomics.

Separation and Comparison of Fountain Pen Inks by Capillary Zone Electrophoresis

Abstract The analysis of inks as part of the detection of fraudulent documents is a small but important part in the operation of a forensic laboratory. Apart from optical methods, multiple thin layer chromatography (TLC) is used to separate, compare and distinguish inks based on their dye composition. Capillary electrophoresis (CE), a relatively young separation technique with very high resolution power, was used for the analysis of water soluble fountain pen inks. Inks are complex mixtures of synthetic organic and inorganic dyes, surfactants, resins and other components. The study focused on the optimization of the separation of 10% aqueous solutions of commercially available inks with respect to resolution and analysis time. During the method development process different buffers, organic modifiers and surfactants were tested. Good results were obtained with a 100 mM borate buffer at pH 8.0 containing 20% methanol. The separations were reproducible and led to baseline resolution of almost all components of blue and black fountain pen inks. Electropherograms of 15 inks of various manufacturers and countries of origin showed patterns which were in the most cases distinctly different from each other. Initial studies of the separation of extracts of inks from paper were successful and are reported here as well. Therefore, it was concluded that CE is a powerful tool for the identification of water soluble writing inks.

Progesterone metabolism by rat oral mucosa. III. Participation of granulation tissue and fibroblasts.

The metabolism of progesterone by rat granulation tissue was studied. Experimental granulation tissue was produced by implanting viscose‐cellulose sponges beneath the dorsal skin of female and male rats for 21 days. The granuloma capsules and fibroblasts in the sponges were homogenized, and mitochondrial, microsomal and soluble fractions were prepared with differential centrifugation. The subcellular Preparations were incubated with 4‐14C‐progesterone and NADPH for 30 min at PH 7.4 and 37°C. The metabolites were identified with column and multiple thinlayer chromatography and radioautography and quantitated with liquid scintillation counting. The granulation tissue and fibroblasts showed much less activity in metabolizing progesterone than the gingival tissue of either sex. As reported earlier, gingival tissues contain Δ4‐5α‐ and,Δ4‐5β‐steroid hydrogenases and 3α‐, 3β‐, 20α‐ and 20β‐hydroxysteroid dehydrogenases. The granulation tissue and fibroblasts lack 3 β‐hydroxysteroid dehydrogenase activity. In addition, the fibroblasts show no 20 β‐hydroxysteroid dehydrogenase activity. The enhanced metabolism of progesterone during gingival inflammation is thus probably not due to the formation of granulation tissue.

Inexpensive instrument for programmed multiple development in thin-layer chromatography

A programmed multiple thin-layer chromatography (TLC) developer of new design has been constructed and tested. It is controlled by a programmable delay timer and supported by other common solid-state components. The system is dedicated to ‘‘Mode 1’’ operation and features a hard-wired drying interval. Solvent removal is effected by a cold-air blower and the elution chamber includes a solvent pump. This ensures rapid solvent vapor saturation of the TLC atmosphere after a drying cycle. The instrument is relatively inexpensive and can be constructed with facilities provided by many laboratories.

Steroids in urine of the newborn human.

ABSTRACT A pool of 2.6 1 urine was collected from normal male newborns for the first 5 days of life. After Amberlite XAD-2 column chromatography, enzyme hydrolysis and extraction, the steroid residue was fractionated by celite column chromatography and each fraction purified by multiple thin-layer chromatography. Identification and quantitation was achieved by gas-liquid chromatography and gas-liquid chromatography-mass spectrometry. Progesterone 4.6 μg/l, pregnanediol 729 μg/1, 16α-hydroxyprogesterone 713 μg/1, 16α-hydroxypregnenolone 14,000 μ/1, 16α-hydroxydehydroepiandrosterone 2,350 μg/l, 16-dehydroprogesterone 5.3 μg/1, 16-dehydropregnenolone 174 μg/1. The identification of 16-dehydropregnanolone is indicated. Comparison with findings in cord plasma and amniotic fluid are done and the role of Δ16-C21 steroids in newborn urine is discussed.

Separation of some isomeric ionones and methylionones by multiple thin-layer chromatography

Separation of some isomeric ionones and methylionones by multiple thin-layer chromatography