Enzymatic reactions on thin-layer chromatographic plates. II. Phospholipase A2 hydrolysis of phosphatidylcholine and separation of the products on a single plate

A procedure for the phospholipase A2 hydrolysis of phosphatidylcholine on a thin-layer chromatographic plate and subsequent separation of the products on the same plate is described. A 0.2-0.8-mg amount of Russell's viper venom (phospholipase A2) in 0.2 ml of 0.005 M calcium chloride solution was applied on a 0.5-mm silica gel G plate as a band over which 2-5 mg of egg phosphatidylcholine in 0.2 ml of diethyl ether containing 5% of methanol was evenly applied. After the reaction had proceeded for 15-20 min in a diethyl ether-saturated chamber at 25 degrees, the plate was developed with chloroform-methanol-water (65:25:4). The bands were identified and their contents extracted. The extent of hydrolysis under different reaction conditions was evaluated from the amount of lysophosphatidylcholine formed. Approximately 74.6% (maximum) conversion was obtained within 15 min at 25 degrees using a substrate to enzyme ratio of 4:1. The acyl group distributions in the 1- and 2-positions of hen egg phosphatidylcholine obtained from the gas-liquid chromatographic analysis of the methyl ester corresponding to the lyso and free fatty acid band agreed with those obtained by the method of Wells and Hanahan. The method is also applicable to phosphatidylethanolamine.     

Improved thin-layer chromatographic method for the separation of cholesterol,egg phosphatidylcholine,and their degradation products

Degradation products of egg phosphatidylcholine (EPC) and cholesterol were analyzed with different normal- and reversed-phase thin-layer chromatography (TLC) systems. The best separation, in terms of the highest number of degradation products from both analytes, was obtained with a reversed-phase system, using butanol-methanol-water-96-98% (v/v) acetic acid (40 + 40 + 20 + 4, v/v/v/v) as the mobile phase after overnight saturation at 25 degrees C. A special development technique was used. After a first development, the plate was dried and a second development was performed in the same direction. This method enabled us to separate lysophosphatidylcholine, several free fatty acids and hydroperoxides, and several undefined degradation products of EPC and cholesterol. All products were visualized after the plate was dipped in a 1% (v/v) solution of 4-methoxybenzaldehyde in 98% sulfuric acid-96-98% (v/v) acetic acid-ethanol-water (2 + 10 + 60 + 30), presenting a blue color or a white spot against a colored background. After activation at 110 degrees C, a stable color for both analytes was reached after 12 min. Precision of <5% was obtained at 2 levels of analysis. Good linearity was obtained in the range of 5-30 microg for EPC (r = 0.991) and 5-40 microg for cholesterol (r = 0.991). These results show that TLC can be an inexpensive and easy alternative for the analysis of EPC and cholesterol.     

The separation of arsenic-77 in a carrier-free state from the parent nuclide germanium-77 by a thin-layer chromatographic method

⁷⁷As(III) and⁷⁷As(V) were separated from neutron-irradiated GeO₂ by a thin-layer chromatographic method, in which silica gel was used as adsorbent and a 2∶1 mixture of methanol and 5N HCl as developer. The Rf values of these nuclides were as follows: 0.00 for⁷⁷Ge, 0.50 for⁷⁷As(III) and 0.94 for⁷⁷As(V). The influence of As(III) carrier added before the separation was investigated on the oxidation state of⁷⁷As recoiled from the parent nuclide. The radiochemical purity of⁷⁷As thus separated was more than 99.9% and the activity due to⁷⁷As could easily be eluted with water from the adsorbent, with 93% recovery.     

Application of fluorescence line-narrowing spectroscopy in analytical chemistry: pyrene and 1-fluoropyrene on a thin-layer chromatographic plate

Fluorescence line-narrowed spectra of pyrene and 1-fluoropyrene on a thin-layer chromatographic plate can be obtained by laser excitation at low temperature (T < 50 K). The resulting vibrationally resolved spectra provide highly specific data so that the much alike compounds can be easily discerned. Quantitative determination down to the low nanogram range is possible if an internal standard is employed. In particular, attention is paid to solvent influences on the spectral features.     

In-line respeciation: an ion-exchange ion chromatographic method applied to the separation of degradation products of chemical warfare nerve agents in soil

The natural background of anions encountered when analyzing soil samples by ion chromatography (IC) present significant problems in the separation, detection and quantification of isopropyl methylphosphonic acid (IMPA) and methylphosphonic acid (MPA), the degradation products of sarin, a chemical warfare nerve agent. Using chemically-suppressed IC with conductivity detection, a commercially available ion-exchange column, and an isocratic binary eluent system, IMPA and MPA were determined in aqueous extracts of soil at sub-ppm (μg/g) concentrations without the need for gradient elution or organic solvent eluent modifiers. Common soil anions such as chloride, nitrate, sulfate and phosphate do not interfere with the analysis method due to the composition of the binary eluent allowing for greater mobilization of multivalent anions (e.g., MPA, carbonate, and sulfate) while monovalent anions (e.g., IMPA and nitrate) are relatively unaffected. Carbonate is selectively removed by in-line respeciation to bicarbonate.     

Studies on digitalis. IV. A method for thin-layer chromatographic separation and determination of digitoxin and cardioactive metabolites in human blood and urine.

A thin-layer chromatographic method for the separation of digitoxin and its cardioactive metabolites in one system is described. Pre-coated silica gel plates impregnated with 15% formamide solution in acetone were developed twice in the same direction (running distance 18cm) with ethyl methyl ketone-xylene (50:50) as solvent. The system showed no border-zone effects, and the reproducibility was good. Samples (5 ml) of serum or urine were extracted with dichloromethane, the extracts were evaporated, the residues were dissolved in 70% ethanol, the ethanol solutions were washed twice with light petroleum and then evaporated, and the residues were dissolved in chloroform-methanol for application to the thin-layer plates. After development, the metabolites were scraped from the plates and analyzed by means of a modified rubidium-86 method. The recovery for the whole procedure was 59%, and the sensitivity of the method permitted the determination of down to 0.5 ng per spot. The method will facilitate the study of digitoxin metabolism in patients undergoing treatment with the drug.     

Application of a simple high-performance liquid chromatographic method for the determination of melphalan in the presence of its hydrolysis products

A procedure for the separation and quantitation of melphalan (L-PAM) and its hydrolysis products by high-performance liquid chromatography is described. The hydrolysis of L-PAM at 25 +/- 0.1 degrees and 41 +/- 0.1 degrees was studied between pH 3.0 and 9.0. The pattern of hydrolysis suggested that L-PAM decomposes via two consecutive pseudo first-order reactions. Pseudo first-order rate constants (k1) were determined for the disappearance of L-PAM at various pH values in buffered solutions and in a formulated product. At both temperatures L-PAM solutions were found to be most stable at low pH. Chloride ion was found to reduce the rate of hydrolysis.     

A quantitative method for the detection of sulfonamide residues in meat and milk samples with a high-performance thin-layer chromatographic method.

Sulfonamides are widely used in veterinary medicine for prophylactic purposes and for the treatment of various infections of food-producing animals. This means that residues of these drugs and their possible metabolites may occur in food of animal origin. In Belgium, a zero tolerance level for sulfonamides in edible animal tissues has been set. In order to check this zero level on a routine basis, a rapid and sensitive method has to be available. For this purpose, a quantitative high-performance thin-layer chromatographic (HPTLC) method for the detection of sulfonamide residues in animal tissue and milk samples has been developed. The sample preparation consists of a liquid extraction followed by a solid phase extraction (SPE) on disposable columns for the meat samples and a matrix solid phase dispersion (MSPD) for the milk samples. A three-multiple development chromatographic system is used for the separation and a derivatization with fluorescamine decreases the minimal detectable quantity per spot from 1.42 to 0.32 ng. The limit of quantification is 4 micrograms/kg for milk and meat samples.     

Polysaccharides of plant tissue cultures. III. Enzymatic hydrolysis of industrial wastes of the biomass of a ginseng tissue culture

Questions of the enzymatic hydrolysis of industrial wastes of the biomass of a ginseng tissue culture by Pektofoetidin P10× (I), Tsellyulaza P10× (II), and Tsellokoningin P10× (III) are discussed. The optimum conditions permitting the hydrolysis of the wastes by the following respective percentages have been selected: (I) 62; (II) 43; (III) 54. Subsequent treatment of the wastes with mineral acids raises the total degree of hydrolysis to 84–88%. The monocarbohydrate compositions of the hydrolysates have been studied by chromatographic methods. Student I. B. Smirnova took part in the work.     

Polysaccharides of plant tissue cultures. III. Enzymatic hydrolysis of industrial wastes of the biomass of a ginseng tissue culture

Questions of the enzymatic hydrolysis of industrial wastes of the biomass of a ginseng tissue culture by Pektofoetidin P10× (I), Tsellyulaza P10× (II), and Tsellokoningin P10× (III) are discussed. The optimum conditions permitting the hydrolysis of the wastes by the following respective percentages have been selected: (I) 62; (II) 43; (III) 54. Subsequent treatment of the wastes with mineral acids raises the total degree of hydrolysis to 84–88%. The monocarbohydrate compositions of the hydrolysates have been studied by chromatographic methods.Student I. B. Smirnova took part in the work.M. V. Frunze Simferopol' State University. Translated from Khimiya Prirodnykh Soedinenii, No. 4, pp. 492–499, July–August, 1988.