Paper Chromatographic Techniques Research Articles

Quantitative determination of oxidized pyridine nucleotides by a paper chromatographic technique

A simplified method for the quantitative determination of oxidized pyridine nucleotides was developed by using a method consisting of a paper chromatographic-technique and a fluorometric technique. By this method, oxidized pyridine nucleotides — NAD + and NADP + — were completely separated and determined. The present method is as accurate as the enzymatic method and is believed to be advantageous for the quantitative determination of oxidized pyridine nucleotides in a large number of samples. The practical limit for the quantitative determination of oxidized pyridine nucleotides was found to be 1 nmole, with a recovery of greater than 95%.

Phosphate esters of human erythrocytes VIII. Separation and identification of hexose diphosphates

The separation of hexose diphosphates by one-dimensional descending paper chromatography has been described. The solvent used consisted of ethanol-ammonium acetate or ethanol-borate mixtures. The methods was used for the separation of glucose, 1,6-diphosphate, fructose 1,6-diphosphate and mannose 1,6-diphosphate from the hexose diphosphate zone obtained after high-voltage paper electrophoresis of protein free filtrates of human erythrocytes. The average concentration of the glucose, fructose and mannose diphosphates in saline washed erythrocytes from fresh human blood were 0.20, 0.13 and 0.19 μmole/g Hb, respectively. In addition some practical aids are described for simplification of paper chromatographic techniques.

Neuere Erfahrungen mit nativer und modifizierter Sorghumstärke

AbstractRecent Experiences on Native and Modified Sorghum Starch. The investigators aimed at characterizing native and modified sorghum starch not only by a number of physicochemical data but also by techniques of paper chromatography in order to gain more insight into the composition of the polysaccharide material. The native starch samples, treated with sodium phosphate and acid and modified by different temperatures yielded useful results on the basis of paper capillary and paper separation analyses which led to a better understanding of their behaviour and enabled further characterization of the sorghum starch material.

Effects of calcium on the keto-acids metabolism of the peanut, Arachis hypogaea

Summary The present study deals with the changes in ketoacids in different plant parts of the peanut ( Arachis hypogaea ) plants as influenced by calcium levels. The plants were grown on acid washed silica sand and the keto-acids were extracted as their 2,4-dinitrophenylhydrazones using freeze dried fresh plant material. Two dimensional ascending paper chromatographic technique has been employed to seperate the various keto-acids present in plants. The major keto-acids present in various parts of peanut plants were α-ketomalonic acid, α-ketoglutaric acid, oxaloacetic acid, pyruvic acid, urea and two unidentified spots. Ketomalonic acid was found to be the major stem keto-acid, while urea was major component in roots; and α-ketoglutaric acid was the dominant keto-acid in leaves. It has been shown that keto-acids in general decrease both at high calcium level, complete calcium deficiency and also sodium chloride treated plants as compared to control plants.

Thin layer chromatographic studies of the pungent principles of Ceylon chillies (Capsicum species)

There are many varieties of cultivated chillies in Ceylon and the pungency of the fruit varies markedly with species, colour and size. A thin layer chromatographic study has been undertaken of the hot principles of Capsicum species found in Ceylon. This study shows that there are three compounds in Capsicum species other than capsaicin (vanillyl amide of isodeeenoic acid) which give similar colour reactions as capsaicin. In Capsicum annuum, variety grossum, the hot principle, capsaicin is not detected by this method but instead, two other compounds are present which in a concentrated extract are responsible for the pungency of the fruit. Major variations in the relative concentrations of those pungent principles as determined semi-quantitatively, in the different varieties of Ceylon chillies have been discussed. Kosuge, Inagaki & Uchare (1) using paper-chromatographic techniques separated two pungent principles from certain Japanese species of Capsicum. One of these compounds was the vanillyl amide of 6 -- iso decenoic acid. By the reduction of noneoic acid, Kosuge, Inagaki & Ino (2) showed that the other pungent principles was the vanillyl amide of 8-methyl nonaoic acid. Jentzch, Pock & Kubelka (3) showed the occurrence of homedihydrocapsaicin and dihydrocapsaicin in Capsicum species. A thin layer chromatographic method was described by Spanyar & Blazovich (4) for the determination of capsaicin in ground paprika. The workers tested more than ten solvent system described in the literature for the separation of capsaicin, and reported that the best solvent system for the separation of capsaicin was chloroform - ethanol (99 : 1 v/v). This paper gives the results of a qualitative study of the hot principles (capsaicins) of Ceylon chillies. Extraction was carried out according to the method of North (5). The capsaicins were extracted with peroxide free diethyl ether. The ethereal extract was treated with purified kerosene and finally partitioned with an aqueous solution of 60% acetone, which

Identification of a pressor polypeptide in human amniotic fluid

It has been suggested that a new, highly active pressor polypeptide can be generated by the incubation of human amniotic fluid. The present report deals with the identification of this polypeptide through separation by gel filtration in Sephadex G-15 and by paper chromatographic techniques. Inhibition of its pressor effect when incubated with antibody against angiotensin I and II was also studied. Separation with Sephadex yielded three peaks of pressor material, while paper chromatography showed this pressor material to have an Rf different from that of angiotensin I or II. Nevertheless, it was concluded that this pressor material is angiotensin, one of its analogues, or a fragment of the polypeptide since its pressor effect was inhibited by antibody against angiotensin. Further, the pressor activity reappeared after destruction of antibody by boiling.

Studies on the nutrition of the genus Pestalotiopsis. II. Chromatographic analysis of the medium during the utilization of various carbohydrates.

The utilization of 17 different carbohydrates by four isolates ofPestalotiopsis was studied by the paper chromatographic technique.

Metabolism of 14 C-pentachlorophenol in the mouse.

After subcutaneous or intraperitoneal injection of 14C‐pentachloro‐phenol (14C‐PCP) in the mouse (specific activity 1.6 μci/mg, dose 15–37 mg/kg body weight) the distribution of the activity in the body was determined by whole‐body autoradiography and by analysis of the individual organs after oxidation to 14CO2. The highest specific activity was found in the gall bladder and its contents, the wall of the stomach fundus, the contents of the gastro‐intestinal tract and the liver. This shows that there is both a gastric and a biliary secretion of PCP and/or its metabolites, and excretion in the faeces. Most of the activity (72–83 %) was excreted in the urine in four days, about half of the dose in 24 hours. Only traces (< 0.05 %) were detected in the expired air. Identification and estimation of metabolites were performed by paper chromatography and isotope dilution techniques. All the urinary activity in the first 24 hours was due to PCP and probably tetrachlorohydroquinone. At least PCP was excreted in both the free and the conjugated form.

Free Amino Acids in the Eggs and Hemolymph of the Grasshopper Melanoplus sanguinipes (Orthoptera: Acrididae). 1. Identification and Quantitative Changes Throughout Development1

This study involved identification by paper chromatographic techniques and quantization of the free amino acids in the blood of the migratory grasshopper, Melanoplus sanguinipes (F.). Differences between sexes, stage of development, and effects of adult aging were considered. The egg yolk of this species of grasshopper contained 26 amino acids. Of interest was the appearance of several amino acids at different periods during embryonic development. Among others, citrulline and ± and ³ aminobutyric acids were found in postdiapause but not in diapause eggs, and methionine was found in diapause but not in postdiapause eggs. Alpha-aminobutyric acid appeared during the middle of postdiapause development and then was absent until the 2nd nymphal stage. Twenty-six amino acids were identified in the blood of nymphal M. sanguinipes . Tryptophan was observed until the 2nd nymphal stage, after which it was not: observed in any subsequent stage of development. Aspartic acid had a similar pattern except that it was in the 4th, perhaps the 5th, nymphal stage and was not found again except in old grasshoppers. Of particular interest was the high concentration of numerous amino acids in the 3rd nymphal instar. Asparagine, proline, alanine, glycine, glutamine and valine, especially, showed increases at this stage. Most amino acids showed a high concentration during the 3rd nymphal stage. Arginine and glutamic acid showed a high concentration at the 2nd nymphal stage. Twenty-six amino acids also were identified in the blood of adult grasshoppers. However, they are not the same 26 amino acids found in the egg yolk or in nymphal grasshoppers. Taurine was found only in adult grasshoppers, and tryptophan was observed only in the egg yolk. There were sex-related qualitative and quantitative differences in the amino acid patterns. These included the presence of methionine sulfone almost exclusively in females, the more frequent occurrence of ³ aminobutyric acid in females, and the alpha isomer in males. Additionally, there was an apparent correlation between the occurrence of methionine sulfone and sulfoxide and egg formulation and/or gonad maturation. A total of ten ninhydrin-positive compounds were not identified.

Metabolism of 14C-histamine in domestic animals. 3. Horse and pig.

AbstractThe metabolism of 14C‐histamine injected i.v. in 3 ruminant species (goat, sheep, and cow) has been studied previously. In the present study isotope dilution‐ and paper chromatographic techniques were used to examine urinary 14C‐metabolites of i.v. injected 14C‐histamine in the horse and the pig. The urine excreted the first 24 hrs after injection accounted for 64–76 % and 81–91 % of the radioactivity injected in horses and pigs, respectively. Less than 2 % of the injected radioactivity could be accounted for in the feces excreted the first week after injection. Known metabolites in the first 24 hr urine on an average accounted for about 94 % and 103 % of the urinary radioactivity in horses and pigs, respectively. Methylation of his‐tamine to 1.4‐methylhistamine and further oxidation to 1.4‐methylimidazoleacetic acid was quantitatively the most important pathway for histamine degradation, accounting on an average for about 63 % and 76 % of the radioactivity in horse and pig urine, respectively. The horse and the pig seemed to metabolize histamine in quite a similar way which again was quite different from that previously found in the ruminants in which oxidative deamination of histamine to imidazoleacetic acid was the only pathway of quantitative importance. As in ruminants histaminol seemed to be a metabolite of i.v. injected histamine accounting for about 2 % of the urinary radioactivity. The specific radioactivity of histamine was about tenfold larger than for 1.4‐methylmidazoleacetic acid, in horses and pigs.

Formation of 1-Methylhydantoin in Whale Meat Caused by Deterioration of Freshness (II)

Creatine was dissolved in an aqueous extract solution prepared from whale meat and mixed with a small portion of a homogenate of whale meat. After adjustment or without adjustment of the initial pH of the mixture, aerobic and anaerobic decompositions of creatine in the resulting mixtures were surveyed by means of a paper chromatographic technique, with special reference to the formation of 3-methylhydantoic acid and 1-methylhy-dantoin.3-Methylhydantoic acid which was thought to be a possible intermediate of the creatine decomposition was not detected in every case. The formation of 1-methylhydantoin was observed only when a mixture, of which the initial pH had been adjusted to 7.3, was incubated under anaerobic conditions. A great quantity of 1-methylhydantoin suddenly appeared in the mixture after 3 days of incubation at 30°C. A marked decrease in quantity of this substance was observed already on the next day, and only acid a trace amount was found in the mixture after 8 days of incubation.

Peak broadening in paper chromatography and related techniques : VII. The contribution of the macroscopic mobile phase velocity profile to peak broadening in paper and thin-layer chromatography

The contribution of the macroscopic mobile phase velocity profile in a paper or thin-layer strip to longitudinal dispersion in chromatography was investigated. For this purpose the dependence of the plate height on the breadth of the paper or thin-layer strip, scanned by the densitometer, was determined using Whatman W2 paper and Camag D cellulose powder. As longitudinal dispersion depends on transversal (convective and diffusive) dispersion, the latter was determined for both chromatographic techniques.

Eli Lilly Lecture: estrogen metabolism and action.

ISHOULD LIKE to consider two related and converging themes of research that have occupied the attention of our laboratory for a number of years. They were both stimulated by the clinical observations of the palliative effects of steroid hormones including estrogens upon certain tumors of the breast and prostate gland. These observations placed in question the previously held view that tumors are wholly autonomous and not subject to control by normal physiological mechanisms. These hormonal effects gave a great impetus to studies of the biochemistry of the steroid hormones, and especially of the estrogens. Perhaps it would be appropriate to indicate the state of the art in those days. It is now difficult to conceive of a time when isotopically labeled steroids could not be purchased at the corner drug store, when paper, thin-layer, gas-liquid and other chromatographic and partition techniques for separation were not yet available and when the most reliable procedure for the detection and estimation of estr...

Paper chromatographic techniques for the determination of cephalothin and desacetylcephalothin in body fluids.

Paper chromatographic procedures may be used to detect cephalothin and its metabolite desacetylcephalothin in urine, plasma, synovial fluid, and cerebrospinal fluid. Protein-bound antibiotics are released from plasma, cerebrospinal fluid, and synovial fluid by dilution with an equal volume of dimethylformamide. Data are presented on the sample preparation, paper chromatographic system, and other specific techniques.

Comparison of competitive protein binding radioassay of cortisol to double isotope dilution and porter silber methods

The competitive protein binding radioassay of corticoids was performed on 4 pools of plasma, and the results compared to those obtained on the same pools by paper chromatography and protein binding, double isotope dilution, and colorimetric (Porter-Silber reaction) techniques. In the low and normal ranges, the colorimetric method was the only one significantly different, i.e., higher. In the high ranges the colorimetric method was significantly higher; while the paper chromatography and protein binding techniques were significantly lower than the protein binding radioassay for corticoids.

Free amino acids in nervous tissue of the common African toad, Bufo regularis boulenger

1. 1. Seventeen free amino acids were detected in the brain and the spinal cord and sixteen in the nerves respectively of Bufo regularis using the paper chromatographic technique. 2. 2. The qualitative and quantitative differences in the composition of amino acids in the three systems are discussed.

Experimental porphyria induced in rats by hexachloro-benzene. A study of the porphyrins excreted by urine

Severe porphyria was induced and maintained for several months in rats by means of daily hexachlorobenzene administration via stomach tube. Urinary porphyrins with 8-, 7-, 6-, 5- and 4-COOH were isolated and studied by column and paper Chromatographic techniques, both qualitative and quantitative, and their absorption spectra and melting points were determined. It was found that the relative excretory pattern was: uroporphyrin > coproporphyrin > phyriaporphyrins > 5-COOH porphyrin > 6-COOH porphyrin. By chemical decarboxylation, all these porphyrins were found to be the type III isomer with only traces of uroporphyrin I and 5-COOH porphyrin I. The predominant porphyrins in the liver were uroporphyrin III and phyriaporphyrin. These findings were compared with those reported in humans intoxicated with the same chemical and with those in patients with hepatic porphyria cutanea tarda. The main differences among these groups were in the isomeric composition of the uroporphyrin, 5-COOH porphyrin and coproporphyrin. Porphyria induced in rats by hexachlorobenzene appears to provide a useful experimental approach for the study of the metabolic changes involved in human porphyria.

Human porphyria cutanea tarda isolation and properties of the urinary porphyrins

Porphyrins with 8-, 7-, 6-, 5-, and 4-COOH were isolated from urine of patients with porphyria cutanea tarda and were studied by column and paper chromatographic techniques both qualitatively and quantitatively, and their absorption spectra and melting points were determined. It was found that the relative excretion pattern was uroporphyrin > coproporphyrin > phyriaporphyrin > 5-COOH porphyrin > 6-COOH porphyrin. Studies of the isomeric composition of these different porphyrins showed that: the uroporphyrin was a mixture of isomers I and III in a 4:1 proportion; phyriaporphyrin and 6-COOH porphyrin were only type III; 5-COOH porphyrin and coproporphyrin were a mixture of isomers I and III.

Paper Chromatographic Techniques for the Determination of Cephalothin and Desacetylcephalothin in Body Fluids

Paper chromatographic procedures may be used to detect cephalothin and its metabolite desacetylcephalothin in urine, plasma, synovial fluid, and cerebrospinal fluid. Protein-bound antibiotics are released from plasma, cerebrospinal fluid, and synovial fluid by dilution with an equal volume of dimethylformamide. Data are presented on the sample preparation, paper chromatographic system, and other specific techniques.

Inhibition of Nitrogen‐Fixing and Nitrifying Bacteria by Seed Plants

Euphorbia suinna occurs in the pioneer weed stage of succession in abandoned fields in several midwestern states of the United States. It was previously found to be very inhibitory to several test strains of nitrogen-fixing and nitrifying bacteria and to several associated seed plants. The present project was concerned wilh the identification of the chief inhibitors produced by that species, using appropriate column and paper chromatographic techniques, and by tests of bacterial inhibition employing the diffusion technique on solid media. Three hydrolyzable tannins were consistently isolated from extracts of the species. All three tannins and purified reagent tannic acid from at least two commercial sources yielded ellagic acid, gallic acid, and glucose on hydrolj'sis by acid or tannase. All yielded one or more additional phenolics which were not identified. The tannins from E. supina were all slightly different from each other and from commercial reagent tannic acid as indicated by different Rf's on paper, elution sequence from polyamide columns, and relative amounts of glucose, ellagic acid and gallic acid produced on hydrolysis.