Rapid biological and chemical analyses of bacterial endotoxins separated by preparative thin-layer chromatography

Methods have been elaborated for rapid location of biologically active bacterial endotoxin components on preparative thin-layer chromatography (tlc) by Limulus lysate clotting assay. The separation of the components from silicic acid is not necessary. Further assays were established for the semiquantitative estimation of endotoxic activites (Shwartzman reactivity, chick embryo lethality, and non-specific tumor resistance enhancement) without complete removal of silicic acid.Quantitative chemical analytical procedures were also elaborated to determine the molar ratios of the biologically active components separated and detected in the above tlc system. These included phosphorus, hexosamine, heptose, 2-keto-3-deoxyoctonate, and long-chain carboxylic acid content measurements. Here again, the chemical determinations were carried out in the presence of silicic acid.     

Separation of phosphoinositides and other phospholipids by two-dimensional thin-layer chromatography

A simple, rapid, two-dimensional TLC system is presented which resolves the four phosphoinositide cycle phospholipids as well as all commonly encountered major and minor phospholipids. Ca2+-free lipid samples are loaded onto silica gel HL plates and developed first in 48:40:7:5 chloroform:methanol:water:concentrated ammonia, and then in 55:25:5 chloroform:methanol:formic acid. The method was applied successfully to human erythrocytes, human platelets, and BL/VL3 murine lymphoma cells.     

Interspecies comparison of brain ganglioside patterns studied by two-dimensional thin-layer chromatography

Abstract Brain gangliosides from four vertebrate classes (fish, amphibia, aves, and mammalia) were studied by patterns generated with the use of two-dimensional thin-layer chromatography. In an effort to retain possible alkali-labile gangliosides, samples were not base-hydrolyzed. Resultant chromatograms revealed complex patterns of the major known gangliosides and a number of minor and trace molecular species previously unresolved. More than 30 resorcinol-positive components were detected in mammalian neural samples. Our data indicate both qualitative and quantitative differences in the chromatogram patterns between the vertebrate classes, but potentially greater qualitative similarity exists between the lower and higher classes than has previously been noted.     

Rapid thin-layer chromatography of various weak analgesics in saliva

A thin-layer chromatographic method was developed to analyze phenacetin, 4-amino-phenazone and N-methyl-4-aminophenazone (5 and 1.25 μg/ml); bucetin (4-ethoxy-β-hydroxybutyranilide), propyphenazone and N-acetyl-4-aminophenazone (2.5 and 1.25 μg/ml); phenazone (15 and 1.25 μg/ml); and N-acetylsalicylamide (0.25 μg/ml). The method was designed to study the bioavailability of different commercial tablets from salivary concentration data.     

Rapid assay for nitric oxide synthase using thin-layer chromatography

A simple, sensitive, and rapid method to determine the nitric oxide synthase (NOS) activity in crude cell extracts has been developed. The method takes advantage of differential migration of arginine and citrulline on silica gel thin-layer chromatography (TLC) with the specified buffer system. We have shown that products obtained by treating [14C]arginine with crude mouse hippocampal homogenate can be separated by methanol precipitation followed by TLC. The separated products of the enzyme reaction can be quantitated by radiometric scanning of the TLC plate or by counting in a scintillation counter. Inhibition of conversion of l-arginine to l-citrulline by NG-monomethyl-l-arginine acetate, a specific inhibitor of NOS, confirmed the NOS assay described in this investigation. This method is versatile and allows rapid simultaneous assay of several samples in a short period of time. Therefore, this assay is very useful for both qualitative and quantitative estimation of NOS activity.     

The alk-1-enyl group content of mammalian myelin phosphoglycerides by quantitative two-dimensional thin-layer chromatography

Myelin phospholipids have been examined by a separation-reaction-separation procedure for two-dimensional thin-layer chromatography on silica gel. After separation in one dimension, alk-1-enyl groups are cleaved by exposure of the plates to HCl fumes. Development in the second dimension quantitatively separates acid-labile and acid-stable phosphoglycerides as well as the aldehydes released from the acid-labile phosphoglycerides. Myelin phospholipids from the central nervous systems of the rhesus monkey, squirrel monkey, ox, and mouse contain 32-36% acid-labile ethanolamine phosphoglycerides (ethanolamine plasmalogens) and 8-14% acid-stable ethanolamine phosphoglycerides. Acid-labile choline and serine phosphoglycerides account for less than 1% of the myelin phospholipids.     

Rapid determination of ubiquinone profiles in Penicillium by reversed phase high performance thin-layer chromatography

Ubiquinone profiles of nine Penicillium strains were shown to be heterogeneous: Each contained ubiquinones Q9, Q10H₂, and Q12, and two P. chrysogenum strains also contained Q10. One of two isolates of the third P. chrysogenum strain lacked Q10H₂. The analytical method (a) used mild extraction, (b) was experimentally undemanding, (c) involved inexpensive equipment, and (d) was rapid. It is the first report of the use of this method with fungi.     

Rapid detection of malto-oligosaccharide-forming bacterial amylases by high performance anion-exchange chromatography

High performance anion-exchange chromatography with pulsed amperometric detection was applied for the rapid analysis of malto-oligosaccharides formed by extracellular enzyme preparations from 49 starch-degrading bacterial strains isolated from soil and compost samples. Malto-oligosaccharide-forming amylases, indicated by a predominant formation of maltohexaose from starch, were produced by enzyme preparations from four of the isolates growing at pH 7.0 and 10.     

Amniotic fluid phospholipid profile determined by two-dimensional thin-layer chromatography as index of fetal lung maturation.

A phospholipid profile, the main features of which were the lecithin/sphingomyelin (L/S) ratio and the presence or absence of phosphatidylglycerol (PG), was determined in amniotic fluid from 188 patients. There was a mature profile (L/S ratio of at least 2 . 0 and detectable PG) in 145 patients, including seven insulin-dependent diabetics, and noe of their babies developed respiratory distress syndrome (RDS). The L/S ratio was less than 2 . 0 and PG absent in 12 patients, nine of whose babies developed RDS, whereas only three small babies (delivered between 28 and 35 weeks because of fulminant pre-eclampsia or severe abruptio placentae) out of 31 developed RDS when the L/S ratio was less than 2 . 0 but PG was present. When amniotic fluid was collected from the vagina only one out of 69 babies developed RDS when PG was present (regardless of the L/S ratio), while all of seven babies developed RDS when PG was absent. It is concluded that the amniotic fluid phospholipid profile, particularly the presence or absence of PG, gives an accurate assessment of fetal lung maturation. The profile may prove a useful adjunct to the management of high-risk pregnancies, especially after premature membrane rupture and perhaps also when the mother is diabetic.     

Separation of adreno-ovarian steroids by thin-layer chromatography

The major adreno-ovarian steroid hormones (progesterone, estrone, 17α-estradiol, 17β-estradiol, estriol, corticosterone, cortisone, and cortisol) have been separated simultaneously on a single TLC plate without recourse to transfer chromatography. The plate was developed successively twice in benzene/ethanol (95:5, v/v) solvent system. It was then sprayed with rhodamine 6G and a line was drawn isolating the already separated least polar and medium-polar steroids (progesterone, estrone, 17α-estradiol, and 17β-estradiol) with the help of ultraviolet light. Then 5 ml methanol per 100 ml solvent in the tank was added and the plate again developed 2–3 times up to the line drawn, when polar steroids (corticosterone, cortisone, cortisol, and estriol) separated out.     

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.     

Rapid separation of phenylthiohydantoin (PTH) amino acids by thin-layer chromatography on polyamide glass plates

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1. A rapid and sensitive method is described for the two-dimensional separation of 24 PTH derivatives by thin-layer chromatography on commercially available polyamide glass plates. This method gives 21 to 22 unique spots by combination of the solvent systems applied.